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Int. J. Mol. Sci., Volume 16, Issue 2 (February 2015) – 125 articles , Pages 2269-4361

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2686 KiB  
Article
Metastatic Melanoma Cells Evade Immune Detection by Silencing STAT1
by JoDi Lynn Osborn and Susanna F. Greer
Int. J. Mol. Sci. 2015, 16(2), 4343-4361; https://doi.org/10.3390/ijms16024343 - 17 Feb 2015
Cited by 30 | Viewed by 10351
Abstract
Transcriptional activation of major histocompatibility complex (MHC) I and II molecules by the cytokine, interferon γ (IFN-γ), is a key step in cell-mediated immunity against pathogens and tumors. Recent evidence suggests that suppression of MHC I and II expression on multiple tumor types [...] Read more.
Transcriptional activation of major histocompatibility complex (MHC) I and II molecules by the cytokine, interferon γ (IFN-γ), is a key step in cell-mediated immunity against pathogens and tumors. Recent evidence suggests that suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, a leading cause of skin cancer-related deaths. Despite growing awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune-mediated elimination remain unknown. Here, we analyze the dysregulation of the Janus kinase (JAK)/STAT pathway and its role in the suppression of MHC II in melanoma cell lines at the radial growth phase (RGP), the vertical growth phase (VGP) and the metastatic phase (MET). While RGP and VGP cells both express MHC II, MET cells lack not only MHC II, but also the critical transcription factors, interferon response factor (IRF) 1 and its upstream activator, signal transducer and activator of transcription 1 (STAT1). Suppression of STAT1 in vitro was also observed in patient tumor samples, suggesting STAT1 silencing as a global mechanism of MHC II suppression and immunoevasion. Full article
(This article belongs to the Section Biochemistry)
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Figure 1

Figure 1
<p>Representation of the progression from healthy melanocyte, to the radial growth phase (RGP), to the vertical growth phase (VGP), to metastatic (MET) melanoma. (<b>A</b>) Healthy melanocytes exist in a fixed ratio to keratinocytes in the basal layer of the epidermis; (<b>B</b>) RGP cells exhibit uncontrolled cell division and over-production of melanin; (<b>C</b>) VGP cells can spread throughout the epidermis and no longer respond to proliferation, inhibiting cytokines; and (<b>D</b>) MET cells are able to break through the basement membrane and enter the circulation.</p> Full article ">Figure 2
<p>Flow cytometric analysis of MHC II cell surface expression in RGP, VGP and MET cells. (<b>A</b>) Cells were treated with IFN-γ for 0 h (blue), 18 h (orange), 24 h (lime green), 48 h (dark green) or 72 h (mauve). Unstained cells (shaded histogram) are shown as a control. Live cells were stained with APC-conjugated antibody against MHC II, were fixed in 2% paraformaldehyde and were then analyzed on a Fortessa flow cytometer. RGP cells demonstrate basal expression of MHC II that increases following IFN-γ stimulation. VGP cells demonstrate less basal expression of MHC II, with an increase upon IFN-γ stimulation comparable to the basal expression seen in radial growth phase (RGP) cells. MET cells lack cell surface expression of MHC II with, or without, stimulation with IFN-γ. The data shown are representative of a minimum of three experimental replicates; (<b>B</b>) Mean fluorescence intensity of the histograms seen in (<b>A</b>) normalized to the isotype control. Data are the average of three experimental replicates.</p> Full article ">Figure 3
<p>(<b>A</b>) Flow cytometric analysis of IFN-γ receptor expression. Cells were treated with IFN-γ for 0 h (blue), 18 h (orange), 24 h (lime green), 48 h (dark green) or 72 h (mauve). Unstained cells (shaded histogram) are shown as a control. Live cells were stained with PE-conjugated antibody against CD119 (IFN-γ R1), were fixed in 2% paraformaldehyde and were then analyzed on a Fortessa flow cytometer. RGP cells express high levels of the IFN-γ receptor as compared to the unstained control. VGP cells express low levels of the IFN-γ receptor with no change throughout 72 h of stimulation. MET cells express varying amounts of IFN-γ receptor throughout 72 h of stimulation. Changes in receptor expression between time points is not statistically significant (<span class="html-italic">p</span> &gt; 0.05). The data shown are representative of a minimum of three experimental replicates; (<b>B</b>) Mean fluorescence intensity of histograms seen in (<b>A</b>) normalized to the isotype control. The data shown are the average of three experimental replicates.</p> Full article ">Figure 4
<p>Western blot analysis of JAK1 and JAK2 expression. Cells were stimulated with IFN-γ for 0, 0.5 or 4 h. Lysates were cleared of cellular debris, and equal concentrations of protein were separated via SDS-PAGE. Proteins were identified by incubating nitrocellulose with antibodies against JAK1 (RGP, VGP, MET; <b>top</b>) or JAK2 (RGP, VGP, MET; <b>middle</b>). β-Actin was used as a loading control (RGP, VGP, MET; <b>bottom</b>). (<b>A</b>–<b>F</b>) JAK1 and JAK2 are constitutively expressed in RGP, VGP and MET cells. The data shown are representative of a minimum of three experimental replicates. The <span class="html-italic">y</span>-axis represents Pixel Total (quantification of A, C, E). NS: Not Significant; * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001</p> Full article ">Figure 5
<p>Western blot analysis of IRF-1 expression. Cells were stimulated with IFN-γ for 0, 0.5 or 4 h. Lysates were cleared of cellular debris, and equal concentrations of protein were separated via SDS-PAGE. Proteins were identified by incubating nitrocellulose with antibodies against IRF-1 (RGP, VGP, MET; <b>top</b>). β-Actin was used as a loading control (RGP, VGP, MET; <b>bottom</b>). (<b>A</b>,<b>B</b>) IRF-1 is expressed in RGP cells following four hours of IFN-γ stimulation; (<b>C</b>,<b>D</b>) In VGP cells, IRF-1 is expressed to a greater extent after four hours of stimulation, compared to RGP; (<b>E</b>,<b>F</b>) MET cells lack IRF-1 expression following 4 h of IFN-γ stimulation. <span class="html-italic">y</span>-axes (<b>B</b>,<b>D</b>,<b>F</b>) represent Total Pixels (Quantification of <b>A</b>,<b>C</b>,<b>E</b>). NS: Not Significant; **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 6
<p>Western blot analysis of STAT1 expression and phosphorylation. Cells were stimulated with IFN-γ for 0, 0.5 or 4 h. Lysates were cleared of cellular debris, and equal concentrations of protein were separated via SDS-PAGE. Proteins were identified by incubating nitrocellulose with antibodies against STAT1 (RGP, VGP, MET; top) or pSTAT1 (RGP, VGP, MET; middle). β-Actin was used as a loading control (RGP, VGP, MET; bottom). (<b>A</b>,<b>B</b>) STAT1 is constitutively expressed and phosphorylated at tyrosine 701 (Y701) following 30 minutes of IFN-γ stimulation in RGP cells; (<b>C</b>,<b>D</b>) STAT1 expression and phosphorylation in VGP cells is similar to RGP; (<b>E</b>,<b>F</b>) STAT1 expression is absent in MET cells. The data shown are representative of at least three experimental replicates. <span class="html-italic">y</span>-axes (<b>B</b>,<b>D</b>,<b>F</b>) represent Total Pixels (Quantification of <b>A</b>,<b>C</b>,<b>E</b>). NS: Not Significant; (<b>G</b>) MET cells + STAT1. MET cells were transfected with 0, 5, 10 µg of Flag-STAT1 for 24 h followed by 48 h of IFN-γ stimulation. Cell surface expression of MHC II was analyzed via flow cytometry; and (<b>H</b>) Expression of Flag-STAT1 following 24 h of incubation with transfection reagent:plasmid complexes. Both 5 µg (light green) and 10 µg (orange) of STAT1 led to restoration of MHC II on the cell surface, compared to non-transfected cells (light blue) and the unstained control (shaded). ** <span class="html-italic">p</span> &lt; 0.005, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 7
<p>Immunofluorescence staining of STAT1 expression in non-metastatic and metastatic melanocytic lesions from patient samples. Tissue microarrays of patient samples were de-waxed, rehydrated and stained for the expression of STAT1 (red) and metastatic melanoma biomarkers (green). Cell nuclei are stained with DAPI (blue). Regions of interest are circled in white on the STAT1 panels. (<b>A</b>) Normal melanocytes show constitutive STAT1 expression; (<b>B</b>) Primary tumors characterized as RGP show a slight decrease in STAT1 expression; (<b>C</b>) VGP melanoma lesions show an increase in melanoma biomarkers, as well as a decrease in STAT1; (<b>D</b>,<b>E</b>) Metastatic melanoma lesions show a marked increase in metastatic melanoma biomarkers, which correlates with a decrease in STAT1. These data show a correlation between biomarkers of metastasis and the decrease in STAT1 expression in patient tumor samples. Data are representative of images taken from approximately 150 patient samples. Scale bars represent 5 µm.</p> Full article ">
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Article
Introducing a Semi-Coated Model to Investigate Antibacterial Effects of Biocompatible Polymers on Titanium Surfaces
by Andreas Winkel, Wibke Dempwolf, Eva Gellermann, Magdalena Sluszniak, Sebastian Grade, Wieland Heuer, Michael Eisenburger, Henning Menzel and Meike Stiesch
Int. J. Mol. Sci. 2015, 16(2), 4327-4342; https://doi.org/10.3390/ijms16024327 - 17 Feb 2015
Cited by 21 | Viewed by 7875
Abstract
Peri-implant infections from bacterial biofilms on artificial surfaces are a common threat to all medical implants. They are a handicap for the patient and can lead to implant failure or even life-threatening complications. New implant surfaces have to be developed to reduce biofilm [...] Read more.
Peri-implant infections from bacterial biofilms on artificial surfaces are a common threat to all medical implants. They are a handicap for the patient and can lead to implant failure or even life-threatening complications. New implant surfaces have to be developed to reduce biofilm formation and to improve the long-term prognosis of medical implants. The aim of this study was (1) to develop a new method to test the antibacterial efficacy of implant surfaces by direct surface contact and (2) to elucidate whether an innovative antimicrobial copolymer coating of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate (VP:DMMEP 30:70) on titanium is able to reduce the attachment of bacteria prevalent in peri-implant infections. With a new in vitro model with semi-coated titanium discs, we were able to show a dramatic reduction in the adhesion of various pathogenic bacteria (Streptococcus sanguinis, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis), completely independently of effects caused by soluble materials. In contrast, soft tissue cells (human gingival or dermis fibroblasts) were less affected by the same coating, despite a moderate reduction in initial adhesion of gingival fibroblasts. These data confirm the hypothesis that VP:DMMEP 30:70 is a promising antibacterial copolymer that may be of use in several clinical applications. Full article
(This article belongs to the Special Issue Antimicrobial Polymers)
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Figure 1

Figure 1
<p>(<b>A</b>) Sketch of titanium disc and coating pattern; (<b>B</b>–<b>D</b>) Secondary ion mass spectrometry images (image size: 500 µm × 500 µm); (<b>B</b>) Total ion image at the border between coated (bright yellow) and uncoated areas; and (<b>C</b>,<b>D</b>) secondary ion mass spectrometry (SIMS) images of the coated area for different ions.</p> Full article ">Figure 2
<p>Adhesion of bacteria on pure (<b>left</b>) and previously covered surfaces (<b>right</b>) without polymer coating (<span class="html-italic">E. coli</span> used as example)—confocal laser scanning microscopy (CLSM) pictures and quantified data. Scale bar = 100 μm.</p> Full article ">Figure 3
<p>Adhesion of different bacterial species (<b>A</b> = <span class="html-italic">E. coli</span>; <b>B</b> = <span class="html-italic">P. aeruginosa</span>; <b>C</b> = <span class="html-italic">S. sanguinis</span>; <b>D</b> = <span class="html-italic">S. mutans</span>; <b>E</b> = <span class="html-italic">S. aureus</span>; <b>F</b> = <span class="html-italic">S. epidermidis</span>) on titanium discs coated with VP:DMMEP 30:70 (<b>right</b>) relative to the uncoated control (<b>left</b>)—CLSM pictures and quantified data (<b>*</b> <span class="html-italic">p</span> &lt; 0.05). Scale bar = 100 μm.</p> Full article ">Figure 3 Cont.
<p>Adhesion of different bacterial species (<b>A</b> = <span class="html-italic">E. coli</span>; <b>B</b> = <span class="html-italic">P. aeruginosa</span>; <b>C</b> = <span class="html-italic">S. sanguinis</span>; <b>D</b> = <span class="html-italic">S. mutans</span>; <b>E</b> = <span class="html-italic">S. aureus</span>; <b>F</b> = <span class="html-italic">S. epidermidis</span>) on titanium discs coated with VP:DMMEP 30:70 (<b>right</b>) relative to the uncoated control (<b>left</b>)—CLSM pictures and quantified data (<b>*</b> <span class="html-italic">p</span> &lt; 0.05). Scale bar = 100 μm.</p> Full article ">Figure 4
<p>Border region between uncoated (left side of each picture) and coated areas (right side of each picture) of titanium discs after seeding with <span class="html-italic">E. coli</span> (<b>A</b>), <span class="html-italic">S. sanguinis</span> (<b>B</b>) and <span class="html-italic">S. aureus</span> (<b>C</b>). Scale bar = 100 μm.</p> Full article ">Figure 5
<p>Quantification and visualization (SEM) of adhered human gingival fibroblasts on titanium discs coated with VP:DMMEP 30:70 (<b>B</b> = 24 h; <b>D</b> = 72 h) in relation to the uncoated control (<b>A</b> = 24 h; <b>C</b> = 72 h)—SEM pictures and quantified data (<b>*</b> <span class="html-italic">p</span> &lt; 0.05). Scale bar = 100 μm.</p> Full article ">Figure 6
<p>Quantification and visualization (SEM) of adhered human dermis fibroblasts on titanium discs coated with VP:DMMEP 30:70 (<b>B</b> = 24 h; <b>D</b> = 72 h) in relation to the uncoated control (<b>A</b> = 24 h; <b>C</b> = 72 h)—SEM pictures and quantified data (<b>*</b> <span class="html-italic">p</span> &lt; 0.05). Scale bar = 100 μm.</p> Full article ">
5104 KiB  
Article
Comprehensive Analysis Suggests Overlapping Expression of Rice ONAC Transcription Factors in Abiotic and Biotic Stress Responses
by Lijun Sun, Lei Huang, Yongbo Hong, Huijuan Zhang, Fengming Song and Dayong Li
Int. J. Mol. Sci. 2015, 16(2), 4306-4326; https://doi.org/10.3390/ijms16024306 - 17 Feb 2015
Cited by 48 | Viewed by 8284
Abstract
NAC (NAM/ATAF/CUC) transcription factors comprise a large plant-specific gene family that contains more than 149 members in rice. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of [...] Read more.
NAC (NAM/ATAF/CUC) transcription factors comprise a large plant-specific gene family that contains more than 149 members in rice. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. However, biological functions for most of the members in the NAC family remain unknown. In this study, microarray data analyses revealed that a total of 63 ONAC genes exhibited overlapping expression patterns in rice under various abiotic (salt, drought, and cold) and biotic (infection by fungal, bacterial, viral pathogens, and parasitic plants) stresses. Thirty-eight ONAC genes exhibited overlapping expression in response to any two abiotic stresses, among which 16 of 30 selected ONAC genes were upregulated in response to exogenous ABA. Sixty-five ONAC genes showed overlapping expression patterns in response to any two biotic stresses. Results from the present study suggested that members of the ONAC genes with overlapping expression pattern may have pleiotropic biological functions in regulation of defense response against different abiotic and biotic stresses, which provide clues for further functional analysis of the ONAC genes in stress tolerance and pathogen resistance. Full article
(This article belongs to the Special Issue Plant Molecular Biology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Overlapping expression of <span class="html-italic">ONAC</span> genes in response to various abiotic stress conditions. (<b>A</b>) Heat map of <span class="html-italic">ONAC</span> genes showing differential expression patterns under at least two abiotic stress conditions. The change values in treated samples <span class="html-italic">vs.</span> corresponding control, shown as (log<sub>2</sub> <sup>(signal intensity in treatment/signal intensity in control)</sup> = log<sub>2</sub><sup>signal intensity in treatment</sup> − log<sub>2</sub><sup>signal intensity in control</sup>), were used for Treeview (<a href="#app1-ijms-16-04306" class="html-app">Supplementary file 2</a>). The color scale of change values is shown at the bottom; (<b>B</b>) Venn diagram represents number of <span class="html-italic">ONAC</span> genes expressed commonly or specifically under given abiotic stress conditions (<span class="html-italic">t</span>-test <span class="html-italic">p</span> &lt; 0.01); (<b>C</b>) Differential expression of 30 selected <span class="html-italic">ONAC</span> genes under drought, salt, and cold conditions, as analyzed by qPCR. Relative expression of each <span class="html-italic">ONAC</span> gene was calculated by comparison with corresponding control. Error bars represent standard errors of the means from three independent biological replicates.</p> Full article ">Figure 2
<p>Differential expressions of 30 selected <span class="html-italic">ONAC</span> genes in response to ABA. Rice seedlings were treated with ABA for 3 h and expression of <span class="html-italic">ONAC</span> genes was analyzed by quantitative RT-PCR. Relative expression for each <span class="html-italic">ONAC</span> gene was calculated by comparison with corresponding control. Error bars represent standard errors of the means from three independent biological replicates.</p> Full article ">Figure 3
<p>Differential expression of <span class="html-italic">ONAC</span> genes in response to infection by <span class="html-italic">M. oryzae.</span> (<b>A</b>) Heat map of <span class="html-italic">ONAC</span> genes showing differential expression patterns after leaf and root infection by <span class="html-italic">M. oryzae</span>. The change values in treated samples <span class="html-italic">vs.</span> corresponding control, shown as (log<sub>2</sub><sup>(signal intensity in treatment/signal intensity in control)</sup> = log<sub>2</sub><sup>signal intensity in treatment</sup> − log<sub>2</sub><sup>signal intensity in control</sup>), were used for Treeview (<a href="#app1-ijms-16-04306" class="html-app">Supplementary file 2</a>). The color scale of change values is shown at the bottom; (<b>B</b>) Expression of 30 selected <span class="html-italic">ONAC</span> genes in response to infection by <span class="html-italic">M. oryzae</span>. Quantitative RT-PCR analysis was performed for samples of the leaves of 2-week-old Yuanfengzao collected at 72 and 96 h after inoculation with <span class="html-italic">M. oryzae</span>. Relative expression of each <span class="html-italic">ONAC</span> gene was calculated by comparison with corresponding control. Error bars represent standard errors of the means from three independent biological replicates.</p> Full article ">Figure 4
<p>Differential expressions of <span class="html-italic">ONAC</span> genes in response to <span class="html-italic">Xoo</span>, <span class="html-italic">Xoc</span> and RSV. (<b>A</b>,<b>B</b>) Heat maps of <span class="html-italic">ONAC</span> genes showing differential expression patterns in rice seedlings after <span class="html-italic">Xoo</span> and <span class="html-italic">Xoc</span> infection and RSV infection, respectively. The change values in treated samples <span class="html-italic">vs.</span> corresponding control, shown as (log<sub>2</sub><sup>(signal intensity in treatment/signal intensity in control)</sup> = log<sub>2</sub><sup>signal intensity in treatment</sup> − log<sub>2</sub><sup>signal intensity in control</sup>), were used for Treeview (<a href="#app1-ijms-16-04306" class="html-app">Supplementary file 2</a>). The color scale of change values is shown at the bottom; (<b>C</b>) Differential expression of 30 selected <span class="html-italic">ONAC</span> genes in rice after RSV infection; (<b>D</b>) Heat Map of <span class="html-italic">ONAC</span> genes showing differential expression patterns after infestation by <span class="html-italic">S. hemonthica</span>. Quantitative RT-PCR analysis was performed and relative expression for each <span class="html-italic">ONAC</span> gene was calculated by comparison with corresponding control. Error bars represent standard errors of the means from three independent biological replicates.</p> Full article ">Figure 5
<p>Overlapping expression of <span class="html-italic">ONAC</span> genes in response to various biotic and abiotic stresses. (<b>A</b>) Heat map of <span class="html-italic">ONAC</span> genes, showing differential expression patterns in response to <span class="html-italic">M. oryzae</span> and <span class="html-italic">S. hermonthica</span>; (<b>B</b>) Venn diagram represents number of <span class="html-italic">ONAC</span> genes expressed commonly or specifically in response to <span class="html-italic">M. oryzae</span> and <span class="html-italic">S. hermonthica</span> (<span class="html-italic">t</span>-test <span class="html-italic">p</span> &lt; 0.01); (<b>C</b>) Heat map of <span class="html-italic">ONAC</span> genes showing differential expression patterns in response to <span class="html-italic">M. oryzae</span>, RSV, <span class="html-italic">Xoo</span>, and <span class="html-italic">Xoc</span>; (<b>D</b>) Venn diagram represents number of <span class="html-italic">ONAC</span> genes expressed commonly or specifically in response to <span class="html-italic">M. oryzae</span>, RSV, <span class="html-italic">Xoo</span>, and <span class="html-italic">Xoc</span> (<span class="html-italic">t</span>-test <span class="html-italic">p</span> &lt; 0.01); (<b>E</b>) Heat map of <span class="html-italic">ONAC</span> genes showing differential expression patterns under abiotic (salt, drought, and cold) and biotic (infection by <span class="html-italic">M. oryzae</span>, <span class="html-italic">Xoo</span>, <span class="html-italic">Xoc</span>, or RSV, or infestation by <span class="html-italic">S. hermonthica</span>) stress; (<b>F</b>) Venn diagram represents number of <span class="html-italic">ONAC</span> genes expressed commonly or specifically in response to biotic and abiotic stresses (<span class="html-italic">t</span>-test <span class="html-italic">p</span> &lt; 0.01). The change values (<b>A</b>,<b>C</b>,<b>E</b>) in treated samples <span class="html-italic">vs.</span> corresponding control, shown as (log<sub>2</sub><sup>(signal intensity in treatment/signal intensity in control)</sup> = log<sub>2</sub><sup>signal intensity in treatment</sup> − log<sub>2</sub><sup>signal intensity in control</sup>), and the relative expression folds were calculated by 2<sup>change values</sup>. Both of them were used for Treeview (<a href="#app1-ijms-16-04306" class="html-app">Supplementary file 2</a>). The color scales of change values and relative expression folds were shown at the bottom.</p> Full article ">Figure 6
<p>Uprooted phylogenetic tree representing relationships among stress-responsive <span class="html-italic">ONAC</span> genes. Full-length protein sequences of 86 <span class="html-italic">ONAC</span> genes (63 <span class="html-italic">ONAC</span> genes showing overlapping expression patterns, several previously reported <span class="html-italic">ONAC</span> genes, and some homologous <span class="html-italic">ONAC</span> genes) were used for multiple alignments by the clustalw2 program and a phylogenic tree was created and visualized using MEGA 6.</p> Full article ">
1621 KiB  
Review
Personalization of the Immunosuppressive Treatment in Renal Transplant Recipients: The Great Challenge in “Omics” Medicine
by Gianluigi Zaza, Simona Granata, Paola Tomei, Alessandra Dalla Gassa and Antonio Lupo
Int. J. Mol. Sci. 2015, 16(2), 4281-4305; https://doi.org/10.3390/ijms16024281 - 17 Feb 2015
Cited by 28 | Viewed by 10944
Abstract
Renal transplantation represents the most favorable treatment for patients with advanced renal failure and it is followed, in most cases, by a significant enhancement in patients’ quality of life. Significant improvements in one-year renal allograft and patients’ survival rates have been achieved over [...] Read more.
Renal transplantation represents the most favorable treatment for patients with advanced renal failure and it is followed, in most cases, by a significant enhancement in patients’ quality of life. Significant improvements in one-year renal allograft and patients’ survival rates have been achieved over the last 10 years primarily as a result of newer immunosuppressive regimens. Despite these notable achievements in the short-term outcome, long-term graft function and survival rates remain less than optimal. Death with a functioning graft and chronic allograft dysfunction result in an annual rate of 3%–5%. In this context, drug toxicity and long-term chronic adverse effects of immunosuppressive medications have a pivotal role. Unfortunately, at the moment, except for the evaluation of trough drug levels, no clinically useful tools are available to correctly manage immunosuppressive therapy. The proper use of these drugs could potentiate therapeutic effects minimizing adverse drug reactions. For this purpose, in the future, “omics” techniques could represent powerful tools that may be employed in clinical practice to routinely aid the personalization of drug treatment according to each patient’s genetic makeup. However, it is unquestionable that additional studies and technological advances are needed to standardize and simplify these methodologies. Full article
(This article belongs to the Special Issue Pharmacogenetics and Personalized Medicine)
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Figure 1
<p>Tissue localization of major polymorphic enzymes involved in metabolism and disposition of immunosuppressive drugs. TAC: Tacrolimus; mTOR-I: mammalian target of rapamycin (mTOR) inhibitors; CsA: Cyclosporin A; MPA: Micophenolic acid; MMF: Mycophenolate mofetil; AZA: Azathioprine.</p> Full article ">Figure 2
<p>Mechanisms of action and targets of immunosuppressive drugs used in renal transplantation. MPA: Micophenolic acid; MMF: Mycophenolate mofetil.</p> Full article ">Figure 3
<p>Prospective employment of pharmacogenetics and pharmacogenomics research strategies.</p> Full article ">
2267 KiB  
Article
β-Hydroxybutyric Sodium Salt Inhibition of Growth Hormone and Prolactin Secretion via the cAMP/PKA/CREB and AMPK Signaling Pathways in Dairy Cow Anterior Pituitary Cells
by Shou-Peng Fu, Wei Wang, Bing-Run Liu, Huan-Min Yang, Hong Ji, Zhan-Qing Yang, Bin Guo, Ju-Xiong Liu and Jian-Fa Wang
Int. J. Mol. Sci. 2015, 16(2), 4265-4280; https://doi.org/10.3390/ijms16024265 - 16 Feb 2015
Cited by 15 | Viewed by 10908
Abstract
β-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL [...] Read more.
β-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows. Full article
(This article belongs to the Collection G Protein-Coupled Receptor Signaling and Regulation)
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Figure 1

Figure 1
<p>The effect of β-hydroxybutyric acid (BHBA) on mRNA levels of <span class="html-italic">GH</span>, <span class="html-italic">PRL</span> and <span class="html-italic">Pit-1</span> in dairy cow anterior pituitary cells (DCAPCs). (<b>A</b>) The effects of the duration of BHBA treatment on <span class="html-italic">GH</span>, <span class="html-italic">PRL</span>, and <span class="html-italic">Pit</span>-<span class="html-italic">1</span> gene expression; (<b>B</b>) The effects of the dosage of BHBA treatment on the <span class="html-italic">GH</span>, <span class="html-italic">PRL</span>, and <span class="html-italic">Pit-1</span> gene expression; (<b>C</b>) The results of the mRNA levels of <span class="html-italic">GH</span>, <span class="html-italic">PRL</span> and <span class="html-italic">Pit-1</span> in DCAPCs treated with or without prior pertussis toxin (PTX) incubation for 2 h and then stimulated with BHBA for 24 h. <b>*</b> indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the control group, <b>**</b> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>#</sup></b> indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the prior PTX incubation group, <b><sup>##</sup></b> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 2
<p>The effect of BHBA on the secretion of GH and PRL in DCAPCs. (<b>A</b>) The effects of the duration of BHBA treatment on GH and PRL secretion; (<b>B</b>) The effects of the dosage of BHBA treatment on GH and PRL secretion; (<b>C</b>) The results of the secretion levels of GH and PRL in DCAPCs treated with or without prior PTX incubation for 2 h and then stimulated with BHBA for 24 h. <b>*</b> indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the control group, <b>**</b> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>#</sup></b> indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 3
<p>The effect of BHBA on intracellular cAMP levels in DCAPCs. (<b>A</b>) DCAPCs were treated with 2.5 mmol/L BHBA for 0, 0.5, 1.0, 2.0, and 3.0 h; (<b>B</b>) The cells were also treated with or without prior PTX incubation for 2 h and then stimulated with 2.5 mmol/L BHBA for 3 h. * indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the control group, ** indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>##</sup></b> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 4
<p>Effect of The effect of BHBA on the activity of PKA in DCAPCs. ** indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>##</sup></b> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 5
<p>The effect of BHBA on CREB phosphorylation in DCAPCs. (<b>A</b>) The Western blotting results of p-CREB and CREB; (<b>B</b>) The phosphorylation level of CREB. ** indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>##</sup></b> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 6
<p>The effect of BHBA on the mRNA levels of <span class="html-italic">GPR109A</span> and <span class="html-italic">MCT1</span> in DCAPCs. <b>(A</b>) The effect of BHBA on the mRNA levels of <span class="html-italic">GPR109A</span> in DCAPCs; (<b>B</b>) The effect of BHBA on the mRNA levels of <span class="html-italic">MCT1</span> in DCAPCs. * indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the control group.</p> Full article ">Figure 7
<p>The role of AMPK-α in BHBA-regulated GH and PRL transcription and secretion in DCAPCs. (<b>A</b>) The effect of BHBA treatment on the activity of AMPKα in DCAPCs; (<b>B</b>) The effect of BML-275 treatment on the activity of AMPKα induced by BHBA in DCAPCs; (<b>C</b>) The effect of BHBA treatment on the gene expression of <span class="html-italic">GH</span> and <span class="html-italic">PRL</span> in DCAPCs; (<b>D</b>) The effect of BHBA treatment on the secretion level of GH and PRL in DCAPCs; (<b>E</b>) The effect of BML-275 treatment on GH secretion inhibited by BHBA in DCAPCs; (<b>F</b>) The effect of BML-275 treatment on PRL secretion inhibited by BHBA in DCAPCs. HG indicates high glucose (25.0 mM), LG indicates low glucose (5.5 mM), and NG indicates non-glucose (0.0 mM), respectively. * indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the control group, ** indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>#</sup></b> indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the prior PTX incubation group, <sup>##</sup> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 7 Cont.
<p>The role of AMPK-α in BHBA-regulated GH and PRL transcription and secretion in DCAPCs. (<b>A</b>) The effect of BHBA treatment on the activity of AMPKα in DCAPCs; (<b>B</b>) The effect of BML-275 treatment on the activity of AMPKα induced by BHBA in DCAPCs; (<b>C</b>) The effect of BHBA treatment on the gene expression of <span class="html-italic">GH</span> and <span class="html-italic">PRL</span> in DCAPCs; (<b>D</b>) The effect of BHBA treatment on the secretion level of GH and PRL in DCAPCs; (<b>E</b>) The effect of BML-275 treatment on GH secretion inhibited by BHBA in DCAPCs; (<b>F</b>) The effect of BML-275 treatment on PRL secretion inhibited by BHBA in DCAPCs. HG indicates high glucose (25.0 mM), LG indicates low glucose (5.5 mM), and NG indicates non-glucose (0.0 mM), respectively. * indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the control group, ** indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the control group, <b><sup>#</sup></b> indicates <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs.</span> the prior PTX incubation group, <sup>##</sup> indicates <span class="html-italic">p</span> &lt; 0.01 <span class="html-italic">vs.</span> the prior PTX incubation group.</p> Full article ">Figure 8
<p>BHBA mediates GH and PRL gene transcription, translation, and secretion in DCAPCs via the cAMP/PKA/CREB and AMPK signaling pathways. BHBA binds to GPCR and leads to the dissociation of the heterotrimeric G protein complex into G<sub>αi</sub> and G<sub>βγ</sub> subunits. The exchange of GTP from GDP results in the activation of G<sub>αi</sub>, thereby inhibiting adenylyl cyclase (AC) activity. This process results in a decrease in intracellular cAMP levels and a subsequent reduction in PKA activity. The inhibition of PKA activity inhibits CREB phosphorylation, thereby decreasing GH and PRL gene transcription, translation, and secretion directly or indirectly. The A-protomer of PTX penetrates into the host cells and results in the inactivation of G<sub>αi</sub>, which subsequently inhibits the BHBA-mediated signaling pathway. In addition, intracellular BHBA uptake mediated by MCT1 may trigger AMPK signaling and result in the phosphorylation of TSC1-TSC2, leading to a decrease in <span class="html-italic">GH</span> and <span class="html-italic">PRL</span> mRNA translation via mTOR signaling.</p> Full article ">
791 KiB  
Article
Cultivation of Chlorella vulgaris and Arthrospira platensis with Recovered Phosphorus from Wastewater by Means of Zeolite Sorption
by Giorgos Markou, Orily Depraetere, Dries Vandamme and Koenraad Muylaert
Int. J. Mol. Sci. 2015, 16(2), 4250-4264; https://doi.org/10.3390/ijms16024250 - 16 Feb 2015
Cited by 30 | Viewed by 7160
Abstract
In this study, zeolite was employed for the separation and recovery of P from synthetic wastewater and its use as phosphorus (P) source for the cultivation of the green microalga Chlorella vulgaris and the cyanobacterium Arthrospira (Spirulina) platensis. At P-loaded zeolite concentration [...] Read more.
In this study, zeolite was employed for the separation and recovery of P from synthetic wastewater and its use as phosphorus (P) source for the cultivation of the green microalga Chlorella vulgaris and the cyanobacterium Arthrospira (Spirulina) platensis. At P-loaded zeolite concentration of 0.15–1 g/L, in which P was limited, the two species displayed quite different behavior regarding their growth and biomass composition. C. vulgaris preferred to increase the intracellular P and did not synthesize biomass, while A. platensis synthesized biomass keeping the intracellular P as low as possible. In addition under P limitation, C. vulgaris did display some little alteration of the biomass composition, while A. platensis did it significantly, accumulating carbohydrates around 70% from about 15%–20% (control). Both species could desorb P from zeolite biologically. A. platensis could recover over 65% and C. vulgaris 25% of the P bounded onto zeolite. When P-loaded zeolite concentration increased to 5 g/L, P was adequate to support growth for both species. Especially in the case of C. vulgaris, growth was stimulated from the presence of P-loaded zeolite and produced more biomass compared to the control. Full article
(This article belongs to the Special Issue Green Chemistry and the Biorefinery)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Desorption of P from P-loaded zeolite using various desorption solutions. P-loaded zeolite concentration 2.5 g/L, desorption duration 24 h.</p> Full article ">Figure 2
<p>Desorption kinetics of P from P-loaded zeolite into BG-11 and Zarrouk cultivation media. P-loaded zeolite concentration: 2.5 g/L.</p> Full article ">Figure 3
<p>Biomass production during the cultivation of <span class="html-italic">C. vulgaris</span> and <span class="html-italic">A. platensis</span> in cultivation media supplemented with 0.15–1.00 g/L P-loaded zeolite as P source.</p> Full article ">Figure 4
<p>Relation between P-loaded zeolite concentration and maximum biomass density. Controls C1 refers to the culture grown without P-loaded zeolite and without P and controls C2 refers to those that were grown with replete P and without P-loaded zeolite.</p> Full article ">Figure 5
<p>P-recovery of bounded P onto zeolite. Physical/chemical desorption zone refers to the mass of P desorbed from zeolite into control medium without microalgae.</p> Full article ">Figure 6
<p>Intracellular P against concentration of P-loaded zeolite.</p> Full article ">Figure 7
<p>Biomass production during the cultivation of <span class="html-italic">C. vulgaris</span> and <span class="html-italic">A. platensis</span> in cultivation media supplemented with 5 g/L P-loaded zeolite as P source.</p> Full article ">
1532 KiB  
Review
Potential Role of Dipeptidyl Peptidase IV in the Pathophysiology of Heart Failure
by Thiago A. Salles, Leonardo Dos Santos, Valério G. Barauna and Adriana C. C. Girardi
Int. J. Mol. Sci. 2015, 16(2), 4226-4249; https://doi.org/10.3390/ijms16024226 - 16 Feb 2015
Cited by 19 | Viewed by 9494
Abstract
Dipeptidyl peptidase IV (DPPIV) is a widely expressed multifunctional serine peptidase that exists as a membrane-anchored cell surface protein or in a soluble form in the plasma and other body fluids. Numerous substrates are cleaved at the penultimate amino acid by DPPIV, including [...] Read more.
Dipeptidyl peptidase IV (DPPIV) is a widely expressed multifunctional serine peptidase that exists as a membrane-anchored cell surface protein or in a soluble form in the plasma and other body fluids. Numerous substrates are cleaved at the penultimate amino acid by DPPIV, including glucagon-like peptide-1 (GLP-1), brain natriuretic peptide (BNP) and stromal cell-derived factor-1 (SDF-α), all of which play important roles in the cardiovascular system. In this regard, recent reports have documented that circulating DPPIV activity correlates with poorer cardiovascular outcomes in human and experimental heart failure (HF). Moreover, emerging evidence indicates that DPPIV inhibitors exert cardioprotective and renoprotective actions in a variety of experimental models of cardiac dysfunction. On the other hand, conflicting results have been found when translating these promising findings from preclinical animal models to clinical therapy. In this review, we discuss how DPPIV might be involved in the cardio-renal axis in HF. In addition, the potential role for DPPIV inhibitors in ameliorating heart disease is revised, focusing on the effects of the main DPPIV substrates on cardiac remodeling and renal handling of salt and water. Full article
(This article belongs to the Special Issue Pathogenesis of Cardiac Arrhythmias and Heart Failure)
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Figure 1

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<p>Schematic model depicting the possible role of DPPIV in the pathophysiology of HF. Several stimuli may increase the activity and abundance of both soluble and cardiac DPPIV in HF during the acute and/or chronic stages of this syndrome. High DPPIV activity may reduce the biological activity of peptides with cardio-, vaso- and renoprotective actions including glucagon-like peptide-1 (GLP-1), brain natriuretic peptide (BNP), and stromal cell-derived factor-1 α (SDF-1α) leading to poorer cardiovascular outcomes. On the other hand, the protease activity of DPPIV can be beneficial for the cardiovascular system by cleaving neuropeptide Y (NPY) and peptide YY (PYY).</p> Full article ">Figure 2
<p>DPPIV as a therapeutic target in HF. Diagram summarizing the rationale and approaches used to test the hypothesis that DPPIV inhibition may exert cardio and renoprotective effects in experimental and clinical HF as well as the main outcomes obtained in experimental and clinical studies (see text for further detail).</p> Full article ">
2983 KiB  
Article
Phosphoproteomic Analysis of the Highly-Metastatic Hepatocellular Carcinoma Cell Line, MHCC97-H
by Miaomiao Tian, Han Cheng, Zhiqiang Wang, Na Su, Zexian Liu, Changqing Sun, Bei Zhen, Xuechuan Hong, Yu Xue and Ping Xu
Int. J. Mol. Sci. 2015, 16(2), 4209-4225; https://doi.org/10.3390/ijms16024209 - 16 Feb 2015
Cited by 24 | Viewed by 8538
Abstract
Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology [...] Read more.
Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Human Liver Diseases)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Scheme for sample preparation, HPLC multi-IMAC (Immobilized Metal ion Affinity Chromatography) methods and data processing of MHCC97-H (high metastasis) phosphoproteomics.</p> Full article ">Figure 2
<p>Characteristics of the identified unique phosphopeptides in different HPLC fractions and IMAC steps. (<b>A</b>) Distribution of identified unique phosphopeptides in different HPLC fractions; (<b>B</b>) Distribution of identified unique phosphopeptides in different IMAC steps; (<b>C</b>) Distribution of phosphorylated peptides based on their phosphorylation sites (p-sites) in different IMAC steps; (<b>D</b>) The <span class="html-italic">pI</span> value distribution of the identified phosphopeptides in different IMAC steps; (<b>E</b>) The hydrophobicity distribution of the identified phosphopeptides in different IMAC steps.</p> Full article ">Figure 3
<p>Characteristics of the identified unique phosphopeptides in the MHCC97-H cell line. (<b>A</b>) Distribution of phosphopeptides based on their length; (<b>B</b>) Distribution of phosphopeptides depending on their number of p-sites; (<b>C</b>) Distribution of phosphorylation proteins based on their number of p-sites; (<b>D</b>) Distribution of phosphorylation serine (p-Ser), phosphorylation threonine (p-Thr) and phosphorylation tyrosine (p-Tyr) sites in the MHCC97-H cell line protein.</p> Full article ">Figure 4
<p>Analysis of p-sites by sequence motif, Group-based Prediction System (GPS) algorithm with the interaction filter, or <span class="html-italic">in vivo</span> GPS (iGPS), the distribution of amino acid flanking and structural preferences in the MHCC97-H cell line. (<b>A</b>) The sequence motif analysis of p-sites in the MHCC97-H cell line consisting of 14 residues surrounding the targeted site by Motif-X; (<b>B</b>) The top 10 protein kinases with the most p-sites by the prediction of iGPS; (<b>C</b>) The heatmap for the distribution of amino acids flanking p-sites in the MHCC97-H phosphoproteome; (<b>D</b>) The secondary structural distribution for the p-sites.</p> Full article ">Figure 5
<p>Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (top 10) analysis for phosphoproteins. (<b>A</b>) Biological process of GO annotation (top 10); (<b>B</b>) Molecular function of GO annotation (top 10); (<b>C</b>) Cellular component of GO annotation (top 10); (<b>D</b>) The most over-represented KEGG pathways; (<b>E</b>) Comparison of p-sites associated with the spliceosome between MHCC97-H and the normal liver sample; (<b>F</b>) Comparison of phosphoproteins associated with the spliceosome between MHCC97-H and the normal liver sample.</p> Full article ">
1310 KiB  
Review
The Role of BH3-Mimetic Drugs in the Treatment of Pediatric Hepatoblastoma
by Justus Lieber, Sorin Armeanu-Ebinger and Jörg Fuchs
Int. J. Mol. Sci. 2015, 16(2), 4190-4208; https://doi.org/10.3390/ijms16024190 - 16 Feb 2015
Cited by 9 | Viewed by 8664
Abstract
Pediatric hepatoblastoma (HB) is commonly treated by neoadjuvant chemotherapy and surgical tumor resection according to international multicenter trial protocols. Complete tumor resection is essential and survival rates up to 95% have now been achieved in those tumors classified as standard-risk HB. Drug resistance [...] Read more.
Pediatric hepatoblastoma (HB) is commonly treated by neoadjuvant chemotherapy and surgical tumor resection according to international multicenter trial protocols. Complete tumor resection is essential and survival rates up to 95% have now been achieved in those tumors classified as standard-risk HB. Drug resistance and occurrence of metastases remain the major challenges in the treatment of HB, especially in high-risk tumors. These conditions urgently require the development of alternative therapeutic strategies. One of those alternatives is the modulation of apoptosis in HB cells. HBs regularly overexpress anti-apoptotic proteins of the Bcl-family in comparison to healthy liver tissue. This fact may contribute to the development of chemoresistance of HB cells. Synthetic small inhibitory molecules with BH3-mimetic effects, such as ABT-737 and obatoclax, enhance the susceptibility of tumor cells to different cytotoxic drugs and thereby affect initiator proteins of the apoptosis cascade via the intrinsic pathway. Besides additive effects on HB cell viability when used in combination with cytotoxic drugs, BH3-mimetics also play a role in preventing metastasation by reducing adhesion and inhibiting cell migration abilities. Presumably, including additive BH3-mimetic drugs into existing therapeutic regimens in HB patients might allow dose reduction of established cytotoxic drugs and thereby associated immanent side effects, while maintaining the antitumor activity. Furthermore, reduction of tumor growth and inhibition of tumor cell dissemination may facilitate complete surgical tumor resection, which is mandatory in this tumor type resulting in improved survival rates in high-risk HB. Currently, there are phase I and phase II clinical trials in several cancer entities using this potential target. This paper reviews the available literature regarding the use of BH3-mimetic drugs as single agents or in combination with chemotherapy in various malignancies and focuses on results in HB cells. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Human Liver Diseases)
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<p>Effects of BH3-mimetic drugs. Overexpression of proteins of the Bcl-family in tumor cells cause capture of Bak and Bax in BH3-binding sites, which prevents initiation of the apoptosis cascade via intrinsic or extrinsic stimuli (<b>A</b>); BH3-mimetic drugs lead to the release of Bak and Bax, which oligomerize and insert as a complex in the outer mitochondrial membrane. This membrane permeabilisation is followed by cytochrome c release and other pro-apoptotic factors into the cytoplasm, initiating apoptosis and leading to cell death (<b>B</b>); BH3-mimetic drugs can enhance dead signals from immune cells (TRAIL, TNF-α) and can influence migration of tumor cells (<b>C</b>).</p> Full article ">
708 KiB  
Article
Genetic Variant in Interleukin-18 Is Associated with Idiopathic Recurrent Miscarriage in Chinese Han Population
by Jun Yue, Yu Tong, Jing Zhou, Qingqing Liu and Jiyun Yang
Int. J. Mol. Sci. 2015, 16(2), 4180-4189; https://doi.org/10.3390/ijms16024180 - 16 Feb 2015
Cited by 13 | Viewed by 6295
Abstract
Levels of IL-18 were significantly lower in women with recurrent miscarriage (RM) than those without idiopathic RM. IL-18 promoter single nucleotide polymorphisms were previously identified to have an impact on IL18 gene transcription activity and influence the level of IL-18 protein production. The [...] Read more.
Levels of IL-18 were significantly lower in women with recurrent miscarriage (RM) than those without idiopathic RM. IL-18 promoter single nucleotide polymorphisms were previously identified to have an impact on IL18 gene transcription activity and influence the level of IL-18 protein production. The aim of this study was to evaluate whether IL-18 gene polymorphisms are risk factors for idiopathic RM in Chinese Han population. Study subjects comprised of 484 idiopathic RM patients and 468 controls. Three polymorphisms (rs360717, rs187238, rs1946518) in IL-18 gene and serum IL-18 concentrations were assessed. rs187238 variant exhibits significant association with RM in additive and recessive genetic model (additive model p = 1.05 × 10−4, dominant model p = 0.025, recessive model p = 2.43 × 10−5). In contrast, rs360717 and rs1946518 are not significantly associated with RM. Serum IL-18 levels are significantly lower in RM cases than in control (111.98 ± 93.13 versus 148.74 ± 130.51 pg/mL, p = 7.42 × 10−7). There are lower levels of serum IL-18 in rs187238 homozygous mutant (CC) than homozygous wild-type (GG) in this study population, including cases and control groups (98.31 ± 86.46 versus 131.87 ± 115.02 pg/mL, p = 0.015). These results suggest that reduced IL-18 levels and rs187238 variant may contribute to pathogenesis of idiopathic RM in Chinese Han population. Full article
(This article belongs to the Special Issue Human Single Nucleotide Polymorphisms and Disease Diagnostics)
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<p>Comparison of serum IL-18 levels among IL18 genotypes. Serum IL-18 was measured using a human IL-18 enzyme-linked immunosorbent assay. Data were expressed as means ± SD. Statistical differences were performed using Mann-Whitney U test for variance non-homogeneity. (<b>a</b>) Serum IL-18 concentrations in cases and controls; (<b>b</b>) Serum IL-18 levels according to rs187238 genotypes among cases and controls.</p> Full article ">
1360 KiB  
Review
Biological Functions of Thyroid Hormone in Placenta
by Cheng-Yi Chen, Chie-Pein Chen and Kwang-Huei Lin
Int. J. Mol. Sci. 2015, 16(2), 4161-4179; https://doi.org/10.3390/ijms16024161 - 16 Feb 2015
Cited by 30 | Viewed by 8543
Abstract
The thyroid hormone, 3,3,5-triiodo-l-thyronine (T3), modulates several physiological processes, including cellular growth, differentiation, metabolism, inflammation and proliferation, via interactions with thyroid hormone response elements (TREs) in the regulatory regions of target genes. Infection and inflammation are critical processes in placental development [...] Read more.
The thyroid hormone, 3,3,5-triiodo-l-thyronine (T3), modulates several physiological processes, including cellular growth, differentiation, metabolism, inflammation and proliferation, via interactions with thyroid hormone response elements (TREs) in the regulatory regions of target genes. Infection and inflammation are critical processes in placental development and pregnancy-related diseases. In particular, infection is the leading cause of neonatal mortality and morbidity worldwide. However, to date, no successful approach has been developed for the effective diagnosis of infection in preterm infants. Pre-eclampsia (PE) is a serious disorder that adversely affects ~5% of human pregnancies. Recent studies identified a multiprotein complex, the inflammasome, including the Nod-like receptor (NLR) family of cytosolic pattern recognition receptors, the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1, which plays a vital role in the placenta. The thyroid hormone modulates inflammation processes and is additionally implicated in placental development and disease. Therefore, elucidation of thyroid hormone receptor-regulated inflammation-related molecules, and their underlying mechanisms in placenta, should facilitate the identification of novel predictive and therapeutic targets for placental disorders. This review provides a detailed summary of current knowledge with respect to identification of useful biomarkers and their physiological significance in placenta. Full article
(This article belongs to the Section Biochemistry)
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Figure 1

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<p>Schematic representation of consensus TRE half-sites and TRα and TRβ isoforms. (<b>A</b>) The consensus half-site of TRE is divided into palindrome, direct repeat and inverted palindrome sequences. Each half-site presents different orientations and nucleotide spacing (N: nucleotide; arrows: half-site orientation); (<b>B</b>) TRα1 and TRβ1 are generated by alternative splicing and promoter usage from TRα and TRβ. TR isoforms contain several functional domains, including an amino terminal region (A/B domain), conserved DNA-binding domain (DBD) (C domain), hinge region that links the DBD and ligand-binding domains (LBD) (D domain), and LBD responsible for receptor dimerization (E domain). Functional domains with similar amino acid sequences are depicted in the same color.</p> Full article ">Figure 2
<p>Schematic representation of the inflammasome activation model. Model depicting the regulation of inflammasome activation. Foreign factors, such as DAMP and PAMP, stimulate NLRs, ASC and pro-caspase-1, followed by formation of the inflammasome complex. Subsequently, pro-caspase-1 is cleaved into caspase-1 via activation of the inflammasome complex. Pro-IL-1β is cleaved into mature IL-1β and secreted to the macrophage extracellular matrix. (DAMP: damage-associated molecular patterns, PAMP: pathogen-associated molecular patterns, NLR: Nod-like receptor, ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain, IL-1β: interleukin-1β).</p> Full article ">
820 KiB  
Article
Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity
by Rosana Aparecida Manólio Soares, Simone Mendonça, Luíla Ívini Andrade De Castro, Amanda Caroline Cardoso Corrêa Carlos Menezes and José Alfredo Gomes Arêas
Int. J. Mol. Sci. 2015, 16(2), 4150-4160; https://doi.org/10.3390/ijms16024150 - 16 Feb 2015
Cited by 108 | Viewed by 8821
Abstract
The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. [...] Read more.
The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. Full article
(This article belongs to the Special Issue Bioactive Proteins and Peptides Derived from Food)
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<p>LC-ESI MS/MS chromatogram showing the major components in the <span class="html-italic">M</span><sub>r</sub> 3000 permeate after a multi-enzyme hydrolysis of amaranth protein isolate.</p> Full article ">Figure 2
<p>Mass spectrum of the selected chromatographic peaks (<span class="html-italic">m</span>/<span class="html-italic">z</span> 232, <span class="html-italic">m</span>/<span class="html-italic">z</span> 288, and <span class="html-italic">m</span>/<span class="html-italic">z</span> 387).</p> Full article ">Figure 3
<p>Evaluation of HMG-CoA reductase activity in control (absence of peptides or drugs), in positive control (in the presence of pravastatin), in hydrolyzed amaranth protein filtered through a 3 KDa <span class="html-italic">M</span>w cut off membrane, and in added of each peptide. Different letters mean significant differences among samples (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
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Article
Isolation and Molecular Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase Genes in Hevea brasiliensis
by Jia-Hong Zhu, Jing Xu, Wen-Jun Chang and Zhi-Li Zhang
Int. J. Mol. Sci. 2015, 16(2), 4136-4149; https://doi.org/10.3390/ijms16024136 - 16 Feb 2015
Cited by 15 | Viewed by 6734
Abstract
Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes [...] Read more.
Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1–7) of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment. These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production. Full article
(This article belongs to the Section Biochemistry)
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<p>Amino acid sequence alignment of <span class="html-italic">HbACS</span> genes. The conserved glutamate residue (E) marked with an arrowhead is involved in substrate specificity. The seven highly conserved regions (I–VII) among all ACC synthases are underlined. Ser residues implicated in calcium-dependent protein kinase (CDPK) and mitogen-activated protein kinase (MPK6) phosphorylation are marked by a black dot and asterisks, respectively.</p> Full article ">Figure 2
<p>Phylogenetic analysis of <span class="html-italic">Hevea</span> and <span class="html-italic">Arabidopsis</span> 1-Aminocyclopropane-1-carboxylic acid synthase (ACS)-like protein sequences. The accession numbers of <span class="html-italic">Arabidopsis</span> ACS-like known proteins in GenBank are listed as follows: AtACS1(NP_191710); AtACS2(Q06402); AtACS4(NP_179866); AtACS5(AAG50098); At-ACS6(T13019); At-ACS7 (AAG48754); AtACS8(AAG50090); AtACS9(AAG48755); AtACS10 (NP_564804); AtACS11(NP_567330); and AtACS12(NP_199982).</p> Full article ">Figure 3
<p>Neighbor-joining phylogenetic tree and intron-exon structures. The phylogenetic tree (part of the left side) was constructed from HbACSs using the MEGA 6.0 program with the NJ method. Intron and exon structural organization of <span class="html-italic">HbACS</span> genes are described on the right side. Introns and exons are represented by black lines and colored boxes, respectively.</p> Full article ">Figure 4
<p><span class="html-italic">HbACS</span> gene expression in various tissues, <span class="html-italic">i.e</span>., roots (R), barks (B), flowers, leaves (Le) and latex (La). Data are means ± standard error calculated from three independent biological replicates.</p> Full article ">Figure 5
<p>Expression of the <span class="html-italic">HbACS</span> genes in the bark after ethrel was applied. Data are means ± standard error calculated from three independent biological replicates.</p> Full article ">
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Article
Genetic Polymorphisms in Estrogen-Related Genes and the Risk of Breast Cancer among Han Chinese Women
by Min-Ying Sun, Hong-Yan Du, An-Na Zhu, Hui-Ying Liang, Gorka Ruiz De Garibay, Fen-Xia Li, Ming Li and Xue-Xi Yang
Int. J. Mol. Sci. 2015, 16(2), 4121-4135; https://doi.org/10.3390/ijms16024121 - 13 Feb 2015
Cited by 18 | Viewed by 6432
Abstract
Exposure to high levels of estrogen is considered an important risk factor for susceptibility to breast cancer. Common polymorphisms in genes that affect estrogen levels may be associated with breast cancer risk, but no comprehensive study has been performed among Han Chinese women. [...] Read more.
Exposure to high levels of estrogen is considered an important risk factor for susceptibility to breast cancer. Common polymorphisms in genes that affect estrogen levels may be associated with breast cancer risk, but no comprehensive study has been performed among Han Chinese women. In the present study, 32 single-nucleotide polymorphisms (SNPs) in estrogen-related genes were genotyped using the MassARRAY IPLEX platform in 1076 Han Chinese women. Genotypic and allelic frequencies were compared between case and control groups. Unconditional logistic regression was used to assess the effects of SNPs on breast cancer risk. Associations were also evaluated for breast cancer subtypes stratified by estrogen receptor (ER) and progesterone receptor (PR) status. Case-control analysis showed a significant relation between heterozygous genotypes of rs700519 and rs2069522 and breast cancer risk (OR = 0.723, 95% CI = 0.541–0.965, p = 0.028 and OR = 1.500, 95% CI = 1.078–2.087, p = 0.016, respectively). Subgroup comparisons revealed that rs2446405 and rs17268974 were related to ER status, and rs130021 was associated with PR status. Our findings suggest that rs700519 and rs2069522 are associated with susceptibility to breast cancer among the Han Chinese population and have a cumulative effect with three other identified SNPs. Further genetic and functional studies are needed to identify additional SNPs, and to elucidate the underlying molecular mechanisms. Full article
(This article belongs to the Special Issue Emerging Classes of Biomarkers for Molecular Diagnostics)
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Review
The Actin Depolymerizing Factor (ADF)/Cofilin Signaling Pathway and DNA Damage Responses in Cancer
by Chun-Yuan Chang, Jyh-Der Leu and Yi-Jang Lee
Int. J. Mol. Sci. 2015, 16(2), 4095-4120; https://doi.org/10.3390/ijms16024095 - 13 Feb 2015
Cited by 46 | Viewed by 17664
Abstract
The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response [...] Read more.
The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy. Full article
(This article belongs to the Special Issue DNA Damage and Repair in Degenerative Diseases 2014)
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<p>Nuclear entry of Cofilin-1 (CFL-1) via various stresses. (<b>A</b>) CFL-1 can bind G-actin and translocate to the nucleus to form actin rods by different exogenous and endogenous stresses. 1. 10% DMSO treatment; 2. Increase of G-actin concentration; 3. Neural degenerative diseases; 4. Heat shock stress; 5. ATP depletion. This action may avoid energy expenditure and promote chromatin remodeling via actin; (<b>B</b>) Enforced expression of CFL-1 can also occur in the nucleus. In this situation, phosphorylated CFL-1 is also detectable in nucleus, while the mechanism of nuclear entry of phospho-CFL-1 remains unclear. Because LIM kinase 1 (LIMK1) and LMK2 are responsible for CFL-1 phosphorylation in various cell types, the subcellular location of these kinases would determine the mechanisms of nuclear accumulation of phosphorylated CFL-1 and regulate actin dynamics in the cytosol and nucleus.</p> Full article ">Figure 2
<p>Effects of different radiation types on DNA damage and cell survivals. Particulate radiations by α-particles or protons belong to densely ionizing radiation type because they can deposit most energy on their tracks. On the other hand, X-rays and γ-rays possess high penetration ability but leave little energy on the traveling track, so that they are sparsely ionizing radiation. Moreover, sparsely ionizing radiation mainly ionizes water to produce free radicals, which are highly electrophilic and prone to capture electrons from biological components. When DNA locates on the track of these two different radiation types, it displays different levels of damage. Densely ionizing radiation causes more double strand breaks than sparsely ionizing radiation, so that DNA repair will be accordingly less efficient. Under the same dosage, the survival fractions of particulate radiation irradiated cells will be lower than that of X-ray or γ-ray irradiated cells.</p> Full article ">Figure 3
<p>DNA damage-induced molecular responses for DNA repair. In response to ionizing radiation, DNA double strand breaks (DSB) and chromatin relaxation surrounding DSB sites will occur. The MRN complex can sense and bind to DSB sites and quickly recruit ATM kinase for autophosphorylation and paraphosphorylation to various molecules, including NBS1 and Rad50 in the MRN complex. In addition, chromatin relaxation will activate PARP1 to execute PARylation on chromatin associated molecules and ATM kinase. Activated ATM needs to be maintained by acetylation via HAT Tip60, which is negatively regulated by the ATF2-Cul3 protein degradation pathway. Although c-Abl kinase can activate Tip60 at the DSB site, how DSB interacts with c-Abl is unclear. Additionally, whether MRN complex will regulate Tip60 to sustain ATM activity is unknown. ATM also regulates ATF2 activity, and it is likely to be an autoregulatory mechanism of ATM activity. Activated ATM can phosphorylate SMC1, NBS1, BRCA1, MDC1, 53BP1, and H2AX to localize DSB sites for further DSB repair mechanisms, including HRR and NHEJ.</p> Full article ">Figure 4
<p>Regulatory mechanisms of actin dynamics in response to DNA damage. (<b>A</b>) Radiation-induced actin polymerization triggers the release of JMY from G-actin. JMY then enters the nucleus and binds to p53 for p53-dependent apoptosis; (<b>B</b>) When DNA damage occurs, polymerized actins may allow p53 binding to actin filaments and retain p53 in the cytoplasm. DNA repair would dominate p53-mediated apoptosis under this condition; and (<b>C</b>) DNA damage-mediated actin polymerization would be caused by p53 induction of the Rho/RCOK signaling pathway, followed by activation of LIMK2 to phosphorylate and inactivate cofilin. The actin depolymerization rate will be reduced to maintain polymerized actins.</p> Full article ">Figure 5
<p>The putative effects of over-expressed CFL-1 on DNA repair. (<b>A</b>) Under normal condition, IR-induced DNA damage will trigger the activation of the ATM kinase, which phosphorylates H2AX encompassing DNA damage sites. 53BP1 will recognize γ-H2AX and recruit additional DNA repair molecules to fix the damaged DNA and lead to cell survival; and (<b>B</b>) Over-expression of wild type CFL-1 (phosphorylatable) may enter and locate around DNA. After IR, the ATM kinase remains to be activated, but cannot phosphorylate H2AX. Recruitment of 53BP1 to the γ-H2AX sites fails, so that the DNA repair capacity is impaired and results in cell death.</p> Full article ">
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Review
Autophagy as a Regulatory Component of Erythropoiesis
by Jieying Zhang, Kunlu Wu, Xiaojuan Xiao, Jiling Liao, Qikang Hu, Huiyong Chen, Jing Liu and Xiuli An
Int. J. Mol. Sci. 2015, 16(2), 4083-4094; https://doi.org/10.3390/ijms16024083 - 13 Feb 2015
Cited by 54 | Viewed by 10346
Abstract
Autophagy is a process that leads to the degradation of unnecessary or dysfunctional cellular components and long-lived protein aggregates. Erythropoiesis is a branch of hematopoietic differentiation by which mature red blood cells (RBCs) are generated from multi-potential hematopoietic stem cells (HSCs). Autophagy plays [...] Read more.
Autophagy is a process that leads to the degradation of unnecessary or dysfunctional cellular components and long-lived protein aggregates. Erythropoiesis is a branch of hematopoietic differentiation by which mature red blood cells (RBCs) are generated from multi-potential hematopoietic stem cells (HSCs). Autophagy plays a critical role in the elimination of mitochondria, ribosomes and other organelles during erythroid terminal differentiation. Here, the modulators of autophagy that regulate erythroid differentiation were summarized, including autophagy-related (Atg) genes, the B-cell lymphoma 2 (Bcl-2) family member Bcl-2/adenovirus E1B 19 kDa interacting protein 3-like (Nix/Binp3L), transcription factors globin transcription factor 1 (GATA1) and forkhead box O3 (FoxO3), intermediary factor KRAB-associated protein1 (KAP1), and other modulators, such as focal adhesion kinase family-interacting protein of 200-kDa (FIP200), Ca2+ and 15-lipoxygenase. Understanding the modulators of autophagy in erythropoiesis will benefit the autophagy research field and facilitate the prevention and treatment of autophagy-related red blood cell disorders. Full article
(This article belongs to the Special Issue Mitochondrial Dysfunction in Ageing and Diseases)
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<p>Autophagy-related factors are involved in the regulation of signal pathways in erythroid cells. The mTOR pathway is an important pathway that directly modulates the Ulk1 complex, and the inhibition of mTOR represses autophagy-related processes. Atg7 and Nix/Bnip3L are required for the removal of mitochondria, inducing the conversion of LC3-I to its lipid‑modified form, LC3-II, to promote autophagy. miRNAs can regulate the expressions of key transcriptional components, and Ca<sup>2+</sup> promotes the binding of 15-lipoxygenase to reticulocyte mitochondria.</p> Full article ">
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Review
The 15q11.2 BP1–BP2 Microdeletion Syndrome: A Review
by Devin M. Cox and Merlin G. Butler
Int. J. Mol. Sci. 2015, 16(2), 4068-4082; https://doi.org/10.3390/ijms16024068 - 13 Feb 2015
Cited by 156 | Viewed by 23521
Abstract
Patients with the 15q11.2 BP1–BP2 microdeletion can present with developmental and language delay, neurobehavioral disturbances and psychiatric problems. Autism, seizures, schizophrenia and mild dysmorphic features are less commonly seen. The 15q11.2 BP1–BP2 microdeletion involving four genes (i.e., TUBGCP5, CYFIP1, [...] Read more.
Patients with the 15q11.2 BP1–BP2 microdeletion can present with developmental and language delay, neurobehavioral disturbances and psychiatric problems. Autism, seizures, schizophrenia and mild dysmorphic features are less commonly seen. The 15q11.2 BP1–BP2 microdeletion involving four genes (i.e., TUBGCP5, CYFIP1, NIPA1, NIPA2) is emerging as a recognized syndrome with a prevalence ranging from 0.57%–1.27% of patients presenting for microarray analysis which is a two to four fold increase compared with controls. Review of clinical features from about 200 individuals were grouped into five categories and included developmental (73%) and speech (67%) delays; dysmorphic ears (46%) and palatal anomalies (46%); writing (60%) and reading (57%) difficulties, memory problems (60%) and verbal IQ scores ≤75 (50%); general behavioral problems, unspecified (55%) and abnormal brain imaging (43%). Other clinical features noted but not considered as common were seizures/epilepsy (26%), autism spectrum disorder (27%), attention deficit disorder (ADD)/attention deficit hyperactivity disorder (ADHD) (35%), schizophrenia/paranoid psychosis (20%) and motor delay (42%). Not all individuals with the deletion are clinically affected, yet the collection of findings appear to share biological pathways and presumed genetic mechanisms. Neuropsychiatric and behavior disturbances and mild dysmorphic features are associated with genomic imbalances of the 15q11.2 BP1–BP2 region, including microdeletions, but with an apparent incomplete penetrance and variable expressivity. Full article
(This article belongs to the Special Issue The Identification of the Genetic Components of Autism Spectrum Disorders)
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<p>High resolution ideogram representing chromosome 15 showing location of breakpoints BP1 and BP2 (at 15q11.2 band) and BP3 (at 15q13.1 band) involving HERC2 and position of the non-imprinted genes between BP1 and BP2. The three deletion types involving the 15q11–q13 region (<span class="html-italic">i.e</span>., BP1–BP2, typical type I, typical type II) are represented.</p> Full article ">
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Review
Induced Pluripotent Stem Cells and Their Use in Cardiac and Neural Regenerative Medicine
by Stepanka Skalova, Tereza Svadlakova, Wasay Mohiuddin Shaikh Qureshi, Kapil Dev and Jaroslav Mokry
Int. J. Mol. Sci. 2015, 16(2), 4043-4067; https://doi.org/10.3390/ijms16024043 - 13 Feb 2015
Cited by 15 | Viewed by 10184
Abstract
Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. [...] Read more.
Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson’s, Alzheimer’s and Huntington’s disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges. Full article
(This article belongs to the Special Issue Molecular and Cellular Basis of Regeneration and Tissue Repair)
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<p>Generation of iPSCs and their use in cell transplantation.</p> Full article ">
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Article
Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
by Akinori Kashimura, Masahiro Kimura, Kazuaki Okawa, Hirotaka Suzuki, Atsushi Ukita, Satoshi Wakita, Kana Okazaki, Misa Ohno, Peter O. Bauer, Masayoshi Sakaguchi, Yasusato Sugahara and Fumitaka Oyama
Int. J. Mol. Sci. 2015, 16(2), 4028-4042; https://doi.org/10.3390/ijms16024028 - 13 Feb 2015
Cited by 23 | Viewed by 6684
Abstract
Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein [...] Read more.
Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases. Full article
(This article belongs to the Section Biochemistry)
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<p>Schematic representations of the <span class="html-italic">Escherichia coli</span>-expressed fusion proteins. Mouse AMCase contains an <span class="html-italic">N</span>-terminal catalytic domain (CatD) and a <span class="html-italic">C</span>-terminal chitin-binding domain (CBD). (<b>A</b>) Pre-Protein A-AMCase-V5-His; (<b>B</b>) Pre-Protein A-CatD-V5-His; and (<b>C</b>) Pre-Protein A-V5-His. The newly synthesized recombinant proteins contain the Protein A signal sequence, which is processed during secretion.</p> Full article ">Figure 2
<p>Analysis of the <span class="html-italic">E. coli</span>-expressed fusion proteins. (<b>A</b>) 12.5% SDS-PAGE analysis of the recombinant proteins from the periplasmic fractions (Peri 1) of <span class="html-italic">E. coli</span>. The fusion proteins were purified by IgG Sepharose followed by Ni Sepharose chromatography, electrophoresed (1 μg of protein) and visualized in the gel by Coomassie Brilliant Blue R-250 (CBB); (<b>B</b>) Western blot analysis of the recombinant proteins. 0.1 μg protein separated by SDS-PAGE was transferred to a PVDF membrane. Immunoblot was performed with anti-V5-HRP antibody. PA-AMCase, Protein A-AMCase-V5-His; PA-CatD, Protein A-CatD-V5-His; (<b>C</b>) Comparison of the chitinolytic properties of the CatD with the full-length AMCase in 50 μL reactions using 0.1 M Gly-HCl buffer (pH 2.0) or McIlvaine’s buffer (pH 7.0) at 37 °C for 30 min as described in the Experimental Section.</p> Full article ">Figure 3
<p>Characterization of the chitinolytic activity of <span class="html-italic">E. coli</span>-expressed Protein A-CatD-V5-His. (<b>A</b>) pH profile; (<b>B</b>) temperature profile; (<b>C</b>) pH stability profile and (<b>D</b>) thermostability profile of the chitinase activity for recombinant CatD were measured as described in Experimental Section. The results are presented as percentage of the maximum activity obtained in each series of experiments. Error bars represent the mean ± standard deviation from a single experiment conducted in triplicate.</p> Full article ">Figure 4
<p>Affinity of Protein A-CatD-V5-His to chitin beads. Protein A-AMCase-V5-His, Protein A-CatD-V5-His and Protein A-V5-His were mixed and loaded onto chitin bead columns. Chitin binding assays were performed at pH 2.0 (<b>A</b>) or pH 7.6 (<b>B</b>) as described in the Experimental Section. The bound and unbound fractions were analyzed by Western blot using anti-V5-HRP antibody.</p> Full article ">Figure 5
<p>Degradation products of GlcNAc hexamer, colloidal and crystalline chitin and shrimp shell by CatD. GlcNAc hexamer (<b>A</b>); colloidal (<b>B</b>) and crystalline (<b>C</b>) chitin and shrimp shell (<b>D</b>) were used as a substrate to determine the chitinase activity of <span class="html-italic">E. coli</span>-expressed proteins in 0.1 M Gly-HCl (pH 2.0). Reactions were conducted for 10 min, 1 or 16 h at 37 °C. Shrimp shell (<b>D</b>) was digested for 16 h. The chitin fragments generated by the recombinant proteins were analyzed by fluorophore-assisted carbohydrate electrophoresis [<a href="#B9-ijms-16-04028" class="html-bibr">9</a>,<a href="#B26-ijms-16-04028" class="html-bibr">26</a>]. Chitin oligomers are shown in the left margin as standards. Fluorophore-assisted carbohydrate electrophoresis analysis revealed that Protein A-CatD-V5-His releases primarily <span class="html-italic">N,N'</span>-diacetylchitobiose fragments from chitin.</p> Full article ">
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Article
Multiscale Experimental and Theoretical Investigations of Spin Crossover FeII Complexes: Examples of [Fe(phen)2(NCS)2] and [Fe(PM-BiA)2(NCS)2]
by Samir F. Matar, Philippe Guionneau and Guillaume Chastanet
Int. J. Mol. Sci. 2015, 16(2), 4007-4027; https://doi.org/10.3390/ijms16024007 - 12 Feb 2015
Cited by 16 | Viewed by 8024
Abstract
For spin crossover (SCO) complexes, computation results are reported and confirmed with experiments at multiscale levels of the isolated molecule and extended solid on the one hand and theory on the other hand. The SCO phenomenon which characterizes organometallics based on divalent iron [...] Read more.
For spin crossover (SCO) complexes, computation results are reported and confirmed with experiments at multiscale levels of the isolated molecule and extended solid on the one hand and theory on the other hand. The SCO phenomenon which characterizes organometallics based on divalent iron in an octahedral FeN6-like environment with high spin (HS) and low spin (LS) states involves the LS/HS switching at the cost of small energies provided by temperature, pressure or light, the latter connected with Light-Induced Excited Spin-State Trapping (LIESST) process. Characteristic infra red (IR) and Raman vibration frequencies are computed within density functional theory (DFT) framework. In [Fe(phen)2(NCS)2] a connection of selected frequencies is established with an ultra-fast light-induced LS → HS photoswitching mechanism. In the extended solid, density of state DOS and electron localization function (ELF) are established for both LS and HS forms, leading to characterizion of the compound as an insulator in both spin states with larger gaps for LS configuration, while keeping molecular features in the solid. In [Fe(PM-BiA)2(NCS)2], by combining DFT and classical molecular dynamics, the properties and the domains of existence of the different phases are obtained by expressing the potential energy surfaces in a short range potential for Fe–N interactions. Applying such Fe–N potentials inserted in a classical force field and carrying out molecular dynamics (MD) in so-called “semi-classical MD” calculations, lead to the relative energies of HS/LS configurations of the crystal and to the assessment of the experimental (P, T) phase diagram. Full article
(This article belongs to the Special Issue Chemical Bond and Bonding 2015)
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<p>(<b>a</b>) Schematic representation of trends of energy <span class="html-italic">versus</span> metal-ligand separation in a spin crossover system HS-LS states; (<b>b</b>) Relative volumes of HS and LS states with the 3<span class="html-italic">d</span><sup>6</sup> configuration of Fe<sup>II</sup> in an octahedral field. The larger HS volume is due to the occupation of antibonding e<sub>g</sub>* orbitals; and (<b>c</b>) The molecular structure of [Fe<sup>II</sup>(phen)<sub>2</sub>(NCS)<sub>2</sub>] characterized by two neutral bidentate phen (1,10-phenanthroline) ligands and two charged monodentate NCS<sup>−</sup> ligands.</p> Full article ">Figure 2
<p>[Fe(phen)<sub>2</sub>(NCS)<sub>2</sub>] SCO complex: Projection of the orthorhombic structure containing 4 formula units. The molecular entity as sketched in <a href="#ijms-16-04007-f001" class="html-fig">Figure 1</a>c is highlighted in the circle.</p> Full article ">Figure 3
<p>Schematic temperature variation of molar magnetic susceptibility χ<sub>M</sub> of a SCO compound showing the different types of thermal spin transitions encountered: (<b>a</b>) Gradual; (<b>b</b>) Abrupt; (<b>c</b>) Hysteretic; (<b>d</b>) Multistep; (<b>e</b>) Incomplete; and (<b>f</b>) No transition.</p> Full article ">Figure 4
<p>HS [Fe(phen)<sub>2</sub>(NCS)<sub>2</sub>]: Snapshots of the breathing mode of the molecule at 128 cm<sup>−1</sup> reproducing the experimentally observed mode.</p> Full article ">Figure 5
<p>Electronic Density of states (DOS) of [Fe(phen)<sub>2</sub>(NCS)<sub>2</sub>]: (<b>a</b>) LS; and (<b>b</b>) HS. Energy reference at the top of the valence band (<span class="html-italic">E</span><sub>V</sub>), both varieties being insulators.</p> Full article ">Figure 6
<p>[Fe(phen)<sub>2</sub>(NCS)<sub>2</sub>] SCO complex: The molecular character in the solid state is illustrated by the electron localization function ELF slice dominated by intermolecular blue (zero localization) zones opposite to red intra-molecular zones with ELF = 1. Green areas correspond to free electron gas like medium localization (see text).</p> Full article ">Figure 7
<p>Magnetic properties (molar susceptibility χ<sub>M</sub>) function of temperature in the dark (blue diamond), under 830 nm irradiation (red open diamond) and in temperature in the dark after irradiation (dark green square) of phase I (<b>a</b>) and phase II (<b>b</b>) of [Fe(PM-BiA)<sub>2</sub>(NCS)<sub>2</sub>].</p> Full article ">Figure 8
<p>Calculated Infra Red spectra of [Fe(PM-BiA)<sub>2</sub>(NCS)<sub>2</sub>] in the two spin states.</p> Full article ">Figure 9
<p>Sketch of the molecule of [Fe(PM-BiA)<sub>2</sub>(NCS)<sub>2</sub>] SCO compound with the labeling of the three different nitrogen atoms in the coordination sphere of central Fe.</p> Full article ">Figure 10
<p>[Fe(PM-BiA)<sub>2</sub>(NCS)<sub>2</sub>]: (P, T) phase diagram showing four solid phases: LS (I): filled green circles; HS (I): filled blue triangles; LS (II): empty green circles; HS (II): empty blue triangles). The domains delimitated by straight curves generate two triple points. Experimental points from [<a href="#B30-ijms-16-04007" class="html-bibr">30</a>] are presented for comparison (LS (I): filled red squares; HS (I): filled red down triangles; LS (II): empty red square; HS (II): empty red up triangles).</p> Full article ">Figure 11
<p>Scheme of the multiscale structural effects of the SCO probed by X-ray diffraction including some general trends for Fe(II)N<sub>6</sub> based complexes.</p> Full article ">
2019 KiB  
Article
Effect of Porcine Akirin2 on Skeletal Myosin Heavy Chain Isoform Expression
by Xiaoling Chen, Yanliu Luo, Bo Zhou, Zhiqing Huang, Gang Jia, Guangmang Liu, Hua Zhao, Zhouping Yang and Ruinan Zhang
Int. J. Mol. Sci. 2015, 16(2), 3996-4006; https://doi.org/10.3390/ijms16023996 - 12 Feb 2015
Cited by 10 | Viewed by 5605
Abstract
Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. [...] Read more.
Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway. Full article
(This article belongs to the Section Biochemistry)
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<p>Relative <span class="html-italic">Akirin2</span> mRNA expression in different types of muscle tissues of pigs. Total RNA from slow soleus (SOL), longissimus lumborum (LL) and extensor digitorum longus (EDL) muscles of three healthy Duroc × Landrace × Yorkshire (DLY) pigs was used to perform the real-time quantitative PCR. Samples were performed in duplicate. The amount of <span class="html-italic">Akirin2</span> mRNA was normalized to the amount of <span class="html-italic">pβ-actin</span> mRNA. Data were presented as means ± SE (<span class="html-italic">n</span> = 3), in arbitrary units. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>Effect of pAkirin2 on myoblast differentiation. C2C12 myoblasts were seeded in a 24-well plate at 1 × 10<sup>4</sup> cells/well. The cells were transfected with 0.5 µg/well of the plasmid pcDNA3.1(+)-pAkirin2 or the empty vector pcDNA3.1(+) when they reached ~90% confluence and induced to differentiate for 5 days before analysis. Myosin heavy chain (MHC) expression was analyzed by immunofluorescence microscopy (DAPI staining also shown). The images are representative of the results obtained from two independent experiments. Magnification: ×100.</p> Full article ">Figure 3
<p>Effect of pAkirin2 on MHC isoform expression in C2C12 myotubes. C2C12 myoblasts were cultured and transfected as in <a href="#ijms-16-03996-f002" class="html-fig">Figure 2</a>. Two days after the transfection, the mRNA levels of <span class="html-italic">MHCI</span>, <span class="html-italic">MHCIIa</span> and <span class="html-italic">MHCIIb</span> were determined using real-time quantitative PCR. Samples were performed in duplicate. The amount of <span class="html-italic">MHCI</span>, <span class="html-italic">MHCIIa</span> and <span class="html-italic">MHCIIb</span> mRNA were normalized to the amount of <span class="html-italic">GAPDH</span> mRNA and <span class="html-italic">mβ-actin</span> mRNA. Data were presented as means ± SE (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Effect of pAkirin2 on oxidative muscle fiber gene expression in C2C12 myotubes. C2C12 myoblasts were cultured and transfected as in <a href="#ijms-16-03996-f002" class="html-fig">Figure 2</a>. After a transfection of 4 and 8 days, the mRNA levels of <span class="html-italic">MEF2C</span>, <span class="html-italic">NFATc1</span> and <span class="html-italic">MCIP1.4</span> were determined by real-time quantitative PCR. Samples were performed in duplicate. The amount of <span class="html-italic">MEF2C</span>, <span class="html-italic">NFATc1</span> and <span class="html-italic">MCIP1.4</span> mRNA were normalized to the amount of <span class="html-italic">GAPDH</span> mRNA and <span class="html-italic">mβ-actin</span> mRNA. Data were presented as means ± SE (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
983 KiB  
Communication
Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody
by Andrew Gdowski, Amalendu Ranjan, Anindita Mukerjee and Jamboor Vishwanatha
Int. J. Mol. Sci. 2015, 16(2), 3990-3995; https://doi.org/10.3390/ijms16023990 - 12 Feb 2015
Cited by 33 | Viewed by 7078
Abstract
Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles [...] Read more.
Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells. Full article
(This article belongs to the Special Issue Bioactive Nanoparticles 2014)
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<p>Characterization of anti-Anx A2 antibodies encapsulated within poly(lactic-<span class="html-italic">co</span>-glycolic acid) (PLGA) nanoparticles. (<b>a</b>) Dynamic Light Scattering (DLS) measurement of the size and polydispersity index (PDI) of the nanoparticles; (<b>b</b>) Encapsulation efficiency and drug loading of the nanoparticles; (<b>c</b>) Twelve day cumulative antibody release experiment; and (<b>d</b>) Immunoblot of whole cell lysates from breast cancer cell lines showing functional binding of released AnnexinA2 (AnxA2) antibody from the nanoparticle at 36 kD. The BT474 and SKBR3 cell lines are known to have very low AnxA2 expression. MDAMB231, MCF10A, and MCF10CA1a cell lines are known to have high AnxA2 expression. (Bars represent standard error of the mean, <span class="html-italic">n</span> = 3).</p> Full article ">Figure 2
<p>Immunofluorescence of released anti-AnxA2 antibody from nanoparticle (<b>a</b>) MDAMB 231 cells treated with heat inactivated anti-AnxA2 antibody released from nanoparticle; (<b>b</b>) Lower magnification of MDAMB 231 cells treated with anti-AnxA2 antibody released from nanoparticles; (<b>c</b>) Higher magnification of single MDAMB 231 cell treated with released anti-AnxA2 antibody. Green = Alexa fluor 488 tagged secondary antibody. Blue = DAPI.</p> Full article ">
709 KiB  
Article
Isolation and Cytotoxicity Evaluation of the Chemical Constituents from Cephalantheropsis gracilis
by Chi-Fen Chang, Yu-Lin Hsu, Chao-Ying Lee, Chia-Hua Wu, Yang-Chang Wu and Ta-Hsien Chuang
Int. J. Mol. Sci. 2015, 16(2), 3980-3989; https://doi.org/10.3390/ijms16023980 - 12 Feb 2015
Cited by 52 | Viewed by 5967
Abstract
Cephalantheropsis gracilis afforded five new compounds: cephalanthrin-A (1), cephalanthrin-B (2), cephathrene-A (3), cephathrene-B (4), methyl 2-(aminocarbonyl) phenylcarbamate (5), and 52 known compounds. The structures of the new compounds were determined by spectroscopic analysis. [...] Read more.
Cephalantheropsis gracilis afforded five new compounds: cephalanthrin-A (1), cephalanthrin-B (2), cephathrene-A (3), cephathrene-B (4), methyl 2-(aminocarbonyl) phenylcarbamate (5), and 52 known compounds. The structures of the new compounds were determined by spectroscopic analysis. Among the compounds isolated, tryptanthrin (6), phaitanthrin A (7), cephalinone D (19), and flavanthrin (30) showed significant cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines. Full article
(This article belongs to the Section Biochemistry)
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<p>Structures of five new compounds <b>1</b>–<b>5</b>.</p> Full article ">
930 KiB  
Review
Regulation of Translation Factor EEF1D Gene Function by Alternative Splicing
by Taku Kaitsuka and Masayuki Matsushita
Int. J. Mol. Sci. 2015, 16(2), 3970-3979; https://doi.org/10.3390/ijms16023970 - 12 Feb 2015
Cited by 14 | Viewed by 6988
Abstract
Alternative splicing is an exquisite mechanism that allows one coding gene to have multiple functions. The alternative splicing machinery is necessary for proper development, differentiation and stress responses in a variety of organisms, and disruption of this machinery is often implicated in human [...] Read more.
Alternative splicing is an exquisite mechanism that allows one coding gene to have multiple functions. The alternative splicing machinery is necessary for proper development, differentiation and stress responses in a variety of organisms, and disruption of this machinery is often implicated in human diseases. Previously, we discovered a long form of eukaryotic elongation factor 1Bδ (eEF1Bδ; this long-form eEF1Bδ results from alternative splicing of EEF1D transcripts and regulates the cellular stress response by transcriptional activation, not translational enhancement, of heat-shock responsive genes. In this review, we discuss the molecular function of EEF1D alternative splicing products and the estimated implication of human diseases. Full article
(This article belongs to the Special Issue Pre-mRNA Splicing)
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<p>Schematic illustration of the <span class="html-italic">EEF1D</span> gene and protein products. Short- or long-isoform eEF1Bδ mRNA is expressed, depending on whether exon III is skipped. eEF1Bδ functions as a guanine nucleotide exchange factor for eEF1A and has a crucial role in translation fidelity; eEF1BδL functions as a transcription factor for HSE-containing genes. The numbers of amino acids are shown. Asterisks indicate start or stop codons. eEF1Bδ, eukaryotic elongation factor 1Bδ; eEF1BδL, long isoform of eEF1Bδ; HSE, heat-shock element; mRNA, messenger RNA.</p> Full article ">Figure 2
<p>Comparison of eEF1BδL structural domains between human and rodent orthologs. The nuclear localization signal (NLS), leucine-zipper motif and guanine nucleotide exchange factor (GEF) domain are well conserved between human and rodent orthologs. The numbers of amino acids are shown.</p> Full article ">Figure 3
<p>Alignment of the amino acid sequences of mammalian eEF1BδL and other eukaryotic eEF1Bδ proteins. Within the compared sequences, the blue highlights show primary conserved regions, and the gray highlights show secondary conserved regions. Arrows indicate each <span class="html-italic">N</span>-terminus of the mammalian eEF1BδL and other eukaryotic eEF1Bδ proteins. The nuclear localization signal (NLS), leucine-zipper motif and guanine nucleotide exchange factor (GEF) domain are marked by boxes and text. MUSCLE was used to create the alignment [<a href="#B12-ijms-16-03970" class="html-bibr">12</a>].</p> Full article ">
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Article
Multifunctional Composites of Chiral Valine Derivative Schiff Base Cu(II) Complexes and TiO2
by Yuki Takeshita, Kazuya Takakura and Takashiro Akitsu
Int. J. Mol. Sci. 2015, 16(2), 3955-3969; https://doi.org/10.3390/ijms16023955 - 12 Feb 2015
Cited by 13 | Viewed by 6490
Abstract
We have prepared four new Cu(II) complexes containing valine moieties with imidazole ligands at the fourth coordination sites and examined their photo-induced reactions with TiO2 in order of understanding the reaction mechanisms. Under a nitrogen atmosphere, the intermolecular electron transfer reactions (essentially [...] Read more.
We have prepared four new Cu(II) complexes containing valine moieties with imidazole ligands at the fourth coordination sites and examined their photo-induced reactions with TiO2 in order of understanding the reaction mechanisms. Under a nitrogen atmosphere, the intermolecular electron transfer reactions (essentially supramolecular interactions) of these systems, which resulted in the reduction of Cu(II) species to Cu(I) ones, occurred after UV light irradiation. In this study, we have investigated the conditions of the redox reactions in view of substituent effects of aldehyde moieties. The results of cyclic voltammetry (CV) on an rotating ring-disk electrode (RRDE) suggested that the substitution effects and redox potentials were correlated. Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations were also performed to simulate the UV–Vis and circular dichroism (CD) spectra; the results revealed a reasonably good correlation between the substituent effects and the highest occupied molecular orbitals and the lowest unoccupied molecular orbitals (HOMO-LUMO) gaps associated with the most intense transition bands. In addition, we summarized the substitution effects of Cu(II) complexes for their corresponding UV light-induced reactions. Full article
(This article belongs to the Special Issue Supramolecular Interactions)
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<p>Molecular structures of Cu(II) complexes (<b>1</b>–<b>4</b>). Crystalline solvents (water molecules) are omitted for clarity.</p> Full article ">Figure 2
<p>Crystal structure of <b>1</b> with selected atoms labeled. Hydrogen atoms are omitted for clarity.</p> Full article ">Figure 3
<p>Crystal structures of <b>2</b> with selected atoms labeled. Hydrogen atoms are omitted for clarity.</p> Full article ">Figure 4
<p>Crystal structures of <b>4</b> with selected atoms labeled. Hydrogen atoms are omitted for clarity.</p> Full article ">Figure 5
<p>UV–Vis (<b>top</b>) and CD (<b>bottom</b>) spectra of <b>1</b>–<b>4</b> solutions in methanol.</p> Full article ">Figure 6
<p>UV–Vis spectra (<b>top</b>) and CD spectra (<b>bottom</b>) of <b>1</b> calculated by TD-DFT and determined experimentally for comparison.</p> Full article ">Figure 7
<p>UV–Vis spectra (<b>top</b>) and CD spectra (<b>bottom</b>) of <b>2</b> calculated by TD-DFT and determined experimentally for comparison.</p> Full article ">Figure 7 Cont.
<p>UV–Vis spectra (<b>top</b>) and CD spectra (<b>bottom</b>) of <b>2</b> calculated by TD-DFT and determined experimentally for comparison.</p> Full article ">Figure 8
<p>UV–Vis spectra (<b>top</b>) and CD spectra (<b>bottom</b>) of <b>3</b> calculated by TD-DFT and determined experimentally for comparison.</p> Full article ">Figure 9
<p>UV–Vis spectra (<b>top</b>) and CD spectra (<b>bottom</b>) of <b>4</b> calculated by TD-DFT and determined experimentally for comparison.</p> Full article ">Figure 10
<p>The UV–Vis spectral changes of <b>1</b> in the presence of TiO<sub>2</sub> microparticles subjected to UV irradiation every 10 min.</p> Full article ">Figure 11
<p>The UV–Vis spectral changes of <b>2</b> in the presence of TiO<sub>2</sub> microparticles subjected to UV irradiation every 10 min.</p> Full article ">Figure 12
<p>The UV–Vis spectral changes of <b>3</b> in the presence of TiO<sub>2</sub> microparticles subjected to UV irradiation every 10 min.</p> Full article ">Figure 13
<p>The UV–Vis spectral changes of <b>4</b> in the presence of TiO<sub>2</sub> microparticles subjected to UV irradiation every 10 min.</p> Full article ">
829 KiB  
Review
Tumor Immunotargeting Using Innovative Radionuclides
by Françoise Kraeber-Bodéré, Caroline Rousseau, Caroline Bodet-Milin, Cédric Mathieu, François Guérard, Eric Frampas, Thomas Carlier, Nicolas Chouin, Ferid Haddad, Jean-François Chatal, Alain Faivre-Chauvet, Michel Chérel and Jacques Barbet
Int. J. Mol. Sci. 2015, 16(2), 3932-3954; https://doi.org/10.3390/ijms16023932 - 11 Feb 2015
Cited by 44 | Viewed by 9594
Abstract
This paper reviews some aspects and recent developments in the use of antibodies to target radionuclides for tumor imaging and therapy. While radiolabeled antibodies have been considered for many years in this context, only a few have reached the level of routine clinical [...] Read more.
This paper reviews some aspects and recent developments in the use of antibodies to target radionuclides for tumor imaging and therapy. While radiolabeled antibodies have been considered for many years in this context, only a few have reached the level of routine clinical use. However, alternative radionuclides, with more appropriate physical properties, such as lutetium-177 or copper-67, as well as alpha-emitting radionuclides, including astatine-211, bismuth-213, actinium-225, and others are currently reviving hopes in cancer treatments, both in hematological diseases and solid tumors. At the same time, PET imaging, with short-lived radionuclides, such as gallium-68, fluorine-18 or copper-64, or long half-life ones, particularly iodine-124 and zirconium-89 now offers new perspectives in immuno-specific phenotype tumor imaging. New antibody analogues and pretargeting strategies have also considerably improved the performances of tumor immunotargeting and completely renewed the interest in these approaches for imaging and therapy by providing theranostics, companion diagnostics and news tools to make personalized medicine a reality. Full article
(This article belongs to the Special Issue Frontiers of Radioimmunotherapy)
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<p>The concept of pretargeting with the Affinity Enhancement System: a bispecific antibody, designed to bind by one arm a tumor antigen (e.g., carcinomembryonic antigen) and by the other a hapten (e.g., the indium-diethylene triamine pentaacetic acid (DTPA) complex or the histamine-succinyl-glutamine (HSG) pseudo-peptide), is injected first. It distributes in the whole-body and targets the tumor cells. After an interval of several hours to a few days, the radiolabeled bivalent hapten is injected. It binds rapidly to the tumor. At the tumor cell surface, hapten bivalency induces cooperativity, resulting in very slow release.</p> Full article ">Figure 2
<p>Images recorded in a patient with a carcino-embryonic antigen (CEA)-positive lung carcinoma treated by pretargeted radioimmunotherapy using the TF2 anti-CEA bispecific antibody and the <sup>177</sup>Lu-IMP288 peptide. Image shows a good targeting of the lung tumor.</p> Full article ">Figure 3
<p>Positron emission tomography (PET) in a patient with a relapse of medullary thyroid carcinoma recorded after injection of the TF2 anti-carcino-embryonic antigen (CEA) bispecific antibody and the <sup>68</sup>Ga-IMP-288 peptide. Image shows a good detection of a bone lesion.</p> Full article ">Figure 4
<p>Imaging performed in a patient with a metastatic breast carcinoma. (<b>A</b>) Immuno-PET performed using the TF2 anti-CEA bispecific antibody and the <sup>68</sup>Ga-IMP-288 peptide detects a more diffuse bone marrow involvement that FDG-PET (<b>B</b>).</p> Full article ">
2774 KiB  
Article
Valproic Acid as a Potential Inhibitor of Plasmodium falciparum Histone Deacetylase 1 (PfHDAC1): An in Silico Approach
by Mohamed A. Abdallah Elbadawi, Mohamed Khalid Alhaj Awadalla, Muzamil Mahdi Abdel Hamid, Magdi Awadalla Mohamed and Talal Ahmed Awad
Int. J. Mol. Sci. 2015, 16(2), 3915-3931; https://doi.org/10.3390/ijms16023915 - 11 Feb 2015
Cited by 11 | Viewed by 10782
Abstract
A new Plasmodium falciparum histone deacetylase1 (PfHDAC1) homology model was built based on the highest sequence identity available template human histone deacetylase 2 structure. The generated model was carefully evaluated for stereochemical accuracy, folding correctness and overall structure quality. All evaluations [...] Read more.
A new Plasmodium falciparum histone deacetylase1 (PfHDAC1) homology model was built based on the highest sequence identity available template human histone deacetylase 2 structure. The generated model was carefully evaluated for stereochemical accuracy, folding correctness and overall structure quality. All evaluations were acceptable and consistent. Docking a group of hydroxamic acid histone deacetylase inhibitors and valproic acid has shown binding poses that agree well with inhibitor-bound histone deacetylase-solved structural interactions. Docking affinity dG scores were in agreement with available experimental binding affinities. Further, enzyme-ligand complex stability and reliability were investigated by running 5-nanosecond molecular dynamics simulations. Thorough analysis of the simulation trajectories has shown that enzyme-ligand complexes were stable during the simulation period. Interestingly, the calculated theoretical binding energies of the docked hydroxamic acid inhibitors have shown that the model can discriminate between strong and weaker inhibitors and agrees well with the experimental affinities reported in the literature. The model and the docking methodology can be used in screening virtual libraries for PfHDAC1 inhibitors, since the docking scores have ranked ligands in accordance with experimental binding affinities. Valproic acid calculated theoretical binding energy suggests that it may inhibit PfHDAC1. Full article
(This article belongs to the Section Molecular Recognition)
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<p>Two-dimensional structures of the ligands used in docking work. (<b>a</b>) Trichostatin A (TSA); (<b>b</b>) suberoylanilide hydroxamic acid (SAHA); (<b>c</b>) suberoyl bis-hydroxamic acid (SBHA); and (<b>d</b>) valproic acid.</p> Full article ">Figure 2
<p>Cartoon representation of homologues superposition. Human histone deacetylase 2 (HDAC2) in blue, <span class="html-italic">Plasmodium falciparum</span> histone deacetylase1 (<span class="html-italic">Pf</span>HDAC1) in green and zinc in yellow.</p> Full article ">Figure 3
<p>Verify3D plot of the <span class="html-italic">Plasmodium falciparum</span> histone deacetylase1 <span class="html-italic">Pf</span>HDAC1 homology model.</p> Full article ">Figure 4
<p>Protein Structure Assessment server (ProSA-web) result of the <span class="html-italic">Plasmodium falciparum</span> histone deacetylase1 <span class="html-italic">Pf</span>HDAC1 homology model; the black dot represents the model <span class="html-italic">z</span>-score.</p> Full article ">Figure 5
<p><span class="html-italic">Plasmodium falciparum</span> histone deacetylase1 (<span class="html-italic">Pf</span>HDAC1) active site. (<b>a</b>) Gaussian contour of the <span class="html-italic">Pf</span>HDAC1 model active site (pink represents hydrogen bonding; green represents hydrophobic contact residues; blue represents mild polar amino acids); and (<b>b</b>) key residues of the model active site.</p> Full article ">Figure 6
<p><span class="html-italic">Pf</span>HDAC1 model-ligand interactions. (<b>a</b>) TSA; (<b>b</b>) SAHA; and (<b>c</b>) SBHA. Bond distances are shown in angstroms.</p> Full article ">Figure 7
<p>Valproic acid-<span class="html-italic">Pf</span>HDAC1 complex interactions.</p> Full article ">Figure 8
<p>Root-mean-square deviation (RMSD) of Cα atoms of enzyme-ligand complexes <span class="html-italic">versus</span> time.</p> Full article ">Figure 9
<p>Potential energies (kcal·mol<sup>−1</sup>) of <span class="html-italic">Plasmodium falciparum</span> histone deacetylase1 ligand complexes during molecular dynamics simulation.</p> Full article ">
699 KiB  
Review
Nutritionally Enhanced Food Crops; Progress and Perspectives
by Kathleen L. Hefferon
Int. J. Mol. Sci. 2015, 16(2), 3895-3914; https://doi.org/10.3390/ijms16023895 - 11 Feb 2015
Cited by 144 | Viewed by 26181
Abstract
Great progress has been made over the past decade with respect to the application of biotechnology to generate nutritionally improved food crops. Biofortified staple crops such as rice, maize and wheat harboring essential micronutrients to benefit the world’s poor are under development as [...] Read more.
Great progress has been made over the past decade with respect to the application of biotechnology to generate nutritionally improved food crops. Biofortified staple crops such as rice, maize and wheat harboring essential micronutrients to benefit the world’s poor are under development as well as new varieties of crops which have the ability to combat chronic disease. This review discusses the improvement of the nutritional status of crops to make a positive impact on global human health. Several examples of nutritionally enhanced crops which have been developed using biotechnological approaches will be discussed. These range from biofortified crops to crops with novel abilities to fight disease. The review concludes with a discussion of hurdles faced with respect to public perception, as well as directions of future research and development for nutritionally enhanced food crops. Full article
(This article belongs to the Special Issue Pharmaceuticals and Nutraceuticals by Molecular Farming)
708 KiB  
Article
A Variant in the Osteoprotegerin Gene Is Associated with Coronary Atherosclerosis in Patients with Rheumatoid Arthritis: Results from a Candidate Gene Study
by Cecilia P. Chung, Joseph F. Solus, Annette Oeser, Chun Li, Paolo Raggi, Jeffrey R. Smith and C. Michael Stein
Int. J. Mol. Sci. 2015, 16(2), 3885-3894; https://doi.org/10.3390/ijms16023885 - 11 Feb 2015
Cited by 12 | Viewed by 6407
Abstract
Objective: Patients with rheumatoid arthritis (RA) have accelerated atherosclerosis, but there is limited information about the genetic contribution to atherosclerosis in this population. Therefore, we examined the association between selected genetic polymorphisms and coronary atherosclerosis in patients with RA. Methods: Genotypes for single-nucleotide [...] Read more.
Objective: Patients with rheumatoid arthritis (RA) have accelerated atherosclerosis, but there is limited information about the genetic contribution to atherosclerosis in this population. Therefore, we examined the association between selected genetic polymorphisms and coronary atherosclerosis in patients with RA. Methods: Genotypes for single-nucleotide polymorphisms (SNPs) in 152 candidate genes linked with autoimmune or cardiovascular risk were measured in 140 patients with RA. The association between the presence of coronary artery calcium (CAC) and SNP allele frequency was assessed by logistic regression with adjustment for age, sex, and race. To adjust for multiple comparisons, a false discovery rate (FDR) threshold was set at 20%. Results: Patients with RA were 54 ± 11 years old and predominantly Caucasian (89%) and female (69%). CAC was present in 70 patients (50%). A variant in rs2073618 that encodes an Asn3Lys missense substitution in the osteoprotegerin gene (OPG, TNFRSF11B) was significantly associated with the presence of CAC (OR = 4.09, p < 0.00026) and withstands FDR correction. Conclusion: Our results suggest that a polymorphism of the TNFRSF11B gene, which encodes osteoprotegerin, is associated with the presence of coronary atherosclerosis in patients with RA. Replication of this finding in independent validation cohorts will be of interest. Full article
(This article belongs to the Special Issue Atherosclerosis and Vascular Imaging)
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<p>Prevalence of coronary calcium by genotype. The <span class="html-italic">p</span>-value was obtained using the Fisher’s exact test.</p> Full article ">
873 KiB  
Article
Autism and Intellectual Disability Associated with Mitochondrial Disease and Hyperlactacidemia
by José Guevara-Campos, Lucía González-Guevara and Omar Cauli
Int. J. Mol. Sci. 2015, 16(2), 3870-3884; https://doi.org/10.3390/ijms16023870 - 11 Feb 2015
Cited by 24 | Viewed by 10784
Abstract
Autism spectrum disorder (ASD) with intellectual disability (ID) is a life-long debilitating condition, which is characterized by cognitive function impairment and other neurological signs. Children with ASD-ID typically attain motor skills with a significant delay. A sub-group of ASD-IDs has been linked to [...] Read more.
Autism spectrum disorder (ASD) with intellectual disability (ID) is a life-long debilitating condition, which is characterized by cognitive function impairment and other neurological signs. Children with ASD-ID typically attain motor skills with a significant delay. A sub-group of ASD-IDs has been linked to hyperlactacidemia and alterations in mitochondrial respiratory chain activity. The objective of this report is to describe the clinical features of patients with these comorbidities in order to shed light on difficult diagnostic and therapeutic approaches in such patients. We reported the different clinical features of children with ID associated with hyperlactacidemia and deficiencies in mitochondrial respiratory chain complex II–IV activity whose clinical presentations are commonly associated with the classic spectrum of mitochondrial diseases. We concluded that patients with ASD and ID presenting with persistent hyperlactacidemia should be evaluated for mitochondrial disorders. Administration of carnitine, coenzyme Q10, and folic acid is partially beneficial, although more studies are needed to assess the efficacy of this vitamin/cofactor treatment combination. Full article
(This article belongs to the Special Issue The Identification of the Genetic Components of Autism Spectrum Disorders)
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<p>Magnetic resonance image of patient 2 (<b>A</b>) and 3 (<b>B</b>) showing an increase in lateral cerebral ventricle volume, diffuse enlargement of the cortical spaces, and cortical atrophy.</p> Full article ">
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