International Journal of Molecular Sciences

2479 KiB

Open AccessArticle

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley

by Krisztián Gierczik, Aliz Novák, Mohamed Ahres, András Székely, Alexandra Soltész, Ákos Boldizsár, Zsolt Gulyás, Balázs Kalapos, István Monostori, László Kozma-Bognár, Gábor Galiba and Attila Vágújfalvi

Int. J. Mol. Sci. 2017, 18(8), 1828; https://doi.org/10.3390/ijms18081828 - 22 Aug 2017

Cited by 23 | Viewed by 5695

Abstract

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light—including its spectra—also [...] Read more.

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light—including its spectra—also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeum vulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance. Full article

(This article belongs to the Special Issue Abiotic Stress and Gene Networks in Plants 2017)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
The expression patterns of the HvCCA1 and HvTOC1 (A,B) with white light and low R/FR illumination. In the first two days, the plants were illuminated for 12 h, whereas, in the next two days, they were kept under constant light conditions. Samples were collected every 4 h during four days. Transcript levels were calculated with the ΔCt method. The values on the X-axes show the time in hours after dawn. The white and black bars symbolise the light and dark periods, while grey bars indicate subjective night. The data and error bars, which represent the standard deviation, originated from three technical replicates. Full article ">Figure 2
The expression patterns of HvPITP and HvPI4K genes (A,B) with white light and low R/FR illumination. Conditions are the same as in <a href="#ijms-18-01828-f001" class="html-fig">Figure 1</a>. Full article ">Figure 3
The expression patterns of the HvPLD (put.), HvCBL2, HvCaM.2, HvCDPK12 (A–D) with white light and low R/FR illumination. Conditions are the same as in <a href="#ijms-18-01828-f001" class="html-fig">Figure 1</a>. Full article ">Figure 4
Gene expression patterns of the HvCBF3 and HvCBF6 (A,B) with white light and low R/FR ratio in the spectra. Conditions are the same as in <a href="#ijms-18-01828-f001" class="html-fig">Figure 1</a>. Full article ">Figure 5
Gene expression levels in HvCBF2A, HvCBF4B, HvCBF9 and HvCBF14 (A–D) with white light and low R/FR illumination. Conditions are the same as in <a href="#ijms-18-01828-f001" class="html-fig">Figure 1</a>. Full article ">Figure 6
Graphical summary of our results based on the gene expression analyses. The abbreviations (R/FR, CCA1, LHY, PITP, PI4K, PLC, CBL2, CaM.2, CDPK12 and CAMTA) and the references in the brackets are detailed in the text. Lines ending with arrowheads represent induction, while lines with blunt ends mean inhibition. Full article ">

928 KiB

Open AccessArticle

Systemic Chemokine Levels with “Gut-Specific” Vedolizumab in Patients with Inflammatory Bowel Disease—A Pilot Study

by Stephanie Zwicker, Ronaldo Lira-Junior, Charlotte Höög, Sven Almer and Elisabeth A. Boström

Int. J. Mol. Sci. 2017, 18(8), 1827; https://doi.org/10.3390/ijms18081827 - 22 Aug 2017

Cited by 8 | Viewed by 5300

Abstract

Vedolizumab, a gut-specific biological treatment for inflammatory bowel disease (IBD), is an antibody that binds to the α₄β₇ integrin and blocks T-cell migration into intestinal mucosa. We aimed to investigate chemokine levels in serum of IBD-patients treated with vedolizumab. In [...] Read more.

Vedolizumab, a gut-specific biological treatment for inflammatory bowel disease (IBD), is an antibody that binds to the α₄β₇ integrin and blocks T-cell migration into intestinal mucosa. We aimed to investigate chemokine levels in serum of IBD-patients treated with vedolizumab. In this pilot study, we included 11 IBD patients (8 Crohn’s disease, 3 ulcerative colitis) previously non-respondent to anti-tumor necrosis factor (TNF)-agents. Patients received vedolizumab at week 0, 2 and 6 and were evaluated for clinical efficacy at week 10. Clinical characteristics and routine laboratory parameters were obtained and patients were classified as responders or non-responders. Expression of 21 chemokines in serum was measured using Proximity Extension Assay and related to clinical outcome. At week 10, 6 out of 11 patients had clinically responded. Overall expression of CCL13 increased after treatment. In non-responders, expression of CCL13 and CXCL8 increased after treatment, and CCL20 and CXCL1 expressions were higher compared to responders. In responders, CCL28 decreased after treatment. C-reactive protein (CRP) correlated negatively with 6 chemokines before therapy, but not after therapy. Systemic CCL13 expression increases in IBD-patients after vedolizumab therapy and several chemokine levels differ between responders and non-responders. An increased CCL13-level when starting vedolizumab treatment, might indicate potential prognostic value of measuring chemokine levels when starting therapy with vedolizumab. This study provides new information on modulation of systemic chemokine levels after vedolizumab treatment. Full article

(This article belongs to the Special Issue Dissecting Mechanisms of Action of Biologics in Inflammatory Bowel Diseases)

► Show Figures

Figure 1

Figure 1
Systemic chemokine levels in 11 inflammatory bowel disease-patients previously non-responding to anti-TNF-agents before and at week 10 after induction therapy with vedolizumab. (A) Serum levels of in total 21 chemokines in IBD-patients at baseline (week 0) and after (week 10) treatment with vedolizumab analyzed by multiplexed proximity extension assay (PEA). Serum levels of individual chemokines; (B) CCL13 (MCP-4); (C) CXCL8 (IL-8); (D) CCL20 (LARC); (E) CXCL1 (GRO-α); (F) CCL23 (MPIF-1); (G) CCL28 (MEC) divided by the clinical response into responders and non-responders. Data represent normalized protein expression (NPX) in a log2 scale mean + SEM, * p < 0.05; ** p < 0.01, paired Student’s t-test; Of IBD-patients six were responders and five non-responders. Full article ">Figure 2
Correlations between systemic chemokine levels and clinical parameters in 11 inflammatory bowel disease-patients previously non-responding to anti-TNF-agents before and at week 10 after induction therapy with vedolizumab. Correlations between clinical and routine laboratory parameters with chemokines (A) at baseline (week 0) and (B) after treatment (week 10) with vedolizumab. * p < 0.05, Pearson correlation. Full article ">Figure 3
Chemokine networks in 11 inflammatory bowel disease-patients previously non-responding to anti-TNF-agents before and at week 10 after induction therapy with vedolizumab. Network was built using the correlation coefficient between each pair of chemokines, where connection lines indicate a significant correlation (p < 0.05) between chemokines (A) at baseline (week 0) and (B) after treatment (week 10) with vedolizumab. Each node represents a chemokine. The black line indicates a positive correlation and the red line indicates a negative correlation. Full article ">

17895 KiB

Open AccessArticle

MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells

by Yueh-Hua Chung, Sung-Chou Li, Ying-Hsien Kao, Hao-Lun Luo, Yuan-Tso Cheng, Pey-Ru Lin, Ming-Hong Tai and Po-Hui Chiang

Int. J. Mol. Sci. 2017, 18(8), 1826; https://doi.org/10.3390/ijms18081826 - 22 Aug 2017

Cited by 31 | Viewed by 5679

Abstract

The involvement of microRNAs (miRNAs) in cancer development and their potential as prognostic biomarkers are becoming increasingly known. However, the signature of miRNAs and their regulatory roles in tumorigenesis of upper tract urothelial carcinoma (UTUC) remain to be elucidated. This study aimed to [...] Read more.

The involvement of microRNAs (miRNAs) in cancer development and their potential as prognostic biomarkers are becoming increasingly known. However, the signature of miRNAs and their regulatory roles in tumorigenesis of upper tract urothelial carcinoma (UTUC) remain to be elucidated. This study aimed to profile the miRNA expression pattern in UTUC tumor tissues and identify candidate miRNAs with prognostic and/or therapeutic functions. Methods and Results: We collected 22 UTUC tissue and adjacent normal tissues samples from patients who underwent nephroureterectomy. The miRNAs signatures of three selected UTUC samples using next-generation sequencing showed that miR-30a-5p was significantly downregulated in UTUC tumors compared to adjacent normal tissues. The differentially-expressed miRNAs were specifically validated by quantitative real-time polymerase chain reaction. In addition, the miRNA expression signatures were analyzed with the transcriptome profile characterized by microarray. Further in vitro studies indicated that overexpression of miR-30a-5p significantly suppressed proliferation, migration, and epithelial-to-mesenchymal transition (EMT) in cultured BFTC-909 UTUC cells. As a potential target gene of miR-30a-5p in the tight junction pathway suggested by the pathway enrichment analysis, the reduced expression of tight junction protein claudin-5 in UTUC cells was demonstrated to be upregulated by miR-30a-5p genetic delivery. Conclusions: Taken together, our findings demonstrated that miR-30a-5p inhibits proliferation, metastasis, and EMT, and upregulates the expression of tight junction claudin-5 in UTUC cells. Thus, miR-30a-5p may provide a promising therapeutic strategy for UTUC treatment. Full article

(This article belongs to the Collection Regulation by Non-coding RNAs)

► Show Figures

Graphical abstract

1361 KiB

Open AccessReview

Chemokines as a Conductor of Bone Marrow Microenvironment in Chronic Myeloid Leukemia

by Naofumi Mukaida, Yamato Tanabe and Tomohisa Baba

Int. J. Mol. Sci. 2017, 18(8), 1824; https://doi.org/10.3390/ijms18081824 - 22 Aug 2017

Cited by 27 | Viewed by 8553

Abstract

All blood lineage cells are generated from hematopoietic stem cells (HSCs), which reside in bone marrow after birth. HSCs self-renew, proliferate, and differentiate into mature progeny under the control of local microenvironments including hematopoietic niche, which can deliver regulatory signals in the form [...] Read more.

All blood lineage cells are generated from hematopoietic stem cells (HSCs), which reside in bone marrow after birth. HSCs self-renew, proliferate, and differentiate into mature progeny under the control of local microenvironments including hematopoietic niche, which can deliver regulatory signals in the form of bound or secreted molecules and from physical cues such as oxygen tension and shear stress. Among these mediators, accumulating evidence indicates the potential involvement of several chemokines, particularly CXCL12, in the interaction between HSCs and bone marrow microenvironments. Fusion between breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog (ABL)-1 gene gives rise to BCR-ABL protein with a constitutive tyrosine kinase activity and transforms HSCs and/or hematopoietic progenitor cells (HPCs) into disease-propagating leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). LSCs can self-renew, proliferate, and differentiate under the influence of the signals delivered by bone marrow microenvironments including niche, as HSCs can. Thus, the interaction with bone marrow microenvironments is indispensable for the initiation, maintenance, and progression of CML. Moreover, the crosstalk between LSCs and bone marrow microenvironments can contribute to some instances of therapeutic resistance. Furthermore, evidence is accumulating to indicate the important roles of bone marrow microenvironment-derived chemokines. Hence, we will herein discuss the roles of chemokines in CML with a focus on bone marrow microenvironments. Full article

(This article belongs to the Special Issue Tumor Microenvironment)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Schematic structure of hematopoietic niche. Hematopoietic stem cells (HSCs) are found adjacent to sinusoids and arterioles throughout the bone marrow. In sinusoids, endothelial cells, CXCL12-abundant reticular (CAR) cells, and NestindimLepr+ perivascular cells promote HSC maintenance. NestinbrightNG+ perivascular cells adjacent to arterioles support HSCs. Sympathetic cells contribute to HSC maintenance by directly regulating CXCL12 expression by MSCs. Hematopoiesis can also be regulated by other types of cells such as osteoblasts. Arrows and “T” indicate stimulating and suppressive activities, respesctively. Full article ">Figure 2
Presumed roles of CXCL12 in normal hematopoiesis. CXCL12 is produced by various types of cells in bone marrow, such as endothelial cells, mesenchymal stroma cells (MSCs), and CAR cells. stromal-derived factor (SDF), HSCs are maintained and retained by CXCL12 produced by Lepr+ perivascular cells in sinusoids or NG2+ perivascular cells in arterioles, while lymphoid progenitor cells are maintained by osteoblast-derived CXCL12. In addition, CXCL12 regulates the development of vasculature, which is crucial for hematopoietic functions. Full article ">Figure 3
Presumed roles of CCL3 in chronic myeloid leukemia (CML). A small amount of CCL3 with a potent inhibitory activity for normal HSPCs is produced constitutively by basophils under normal circumstances. In CML bone marrow, a large number of basophil-like leukemia cells are generated from LICs and produce abundantly CCL3, which inhibits normal HSPC proliferation to confer growth advantage to LICs over normal HSPCs. Black arrows indicate the differentiation pathways while the red arrow indicate the competition between normal HSCs and LICs. “T” indicates the suppressive activities. Full article ">

2763 KiB

Open AccessArticle

Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions

by Jianan Shi, Teng Huang, Shuaijie Chai, Yalu Guo, Jian Wei, Shijuan Dou, Liyun Li and Guozhen Liu

Int. J. Mol. Sci. 2017, 18(8), 1822; https://doi.org/10.3390/ijms18081822 - 22 Aug 2017

Cited by 17 | Viewed by 6421

Abstract

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonas reinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD₇₅₀ and total protein contents were evaluated on days 0, 1, 2, [...] Read more.

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonas reinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD₇₅₀ and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatment were identified by calculating the Pearson’s correlation coefficient (PCC) between target protein abundance and total protein content. Histone H3, beta tubulin 1 (TUB-1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL), and mitochondrial F1F0 ATP synthase subunit 6 (ATPs-6) were the top reference proteins, because they were expressed stably under multiple stress conditions. The average relative-fold change (ARF) value of each protein was calculated to identify biomarkers. Heat shock protein 90B (HSP90B), flagellar associated protein (FAP127) and ATP synthase CF0 A subunit (ATPs-A) were suitable biomarkers for multiple treatments, while receptor of activated protein kinase C1 (RCK1), biotin carboxylase (BCR1), mitochondrial phosphate carrier protein (MPC1), and rubisco large subunit N-methyltransferase (RMT1) were suitable biomarkers for the dark, cold, heat, and glucose treatments, respectively. Full article

(This article belongs to the Special Issue Selected Papers from the 6th National Plant Protein Research Congress)

► Show Figures

Figure 1

Figure 1
Culture appearance and cell density of Chlamydomonas reinhardtii grown under abiotic stresses. (A) Photos taken of C. reinhardtii at different time points during culture: control (CK), dark, cold (5 °C), heat (40 °C), NaCl (100 mM), and glucose (1% w/v); (B) Cell number was determined using a hemocytometer under a microscope; (C,D) Optical density of the culture at 750 nm and 680 nm in various treatments. Data are the average values from three independent experiments. Full article ">Figure 2
Total protein contents of Chlamydomonas reinhardtii cells cultured under various abiotic stresses. C. reinhardtii cells were collected by centrifugation at indicated time points, and total proteins were extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (A–C). Coomassie Brilliant Blue-stained gels. (D). Signals extracted from A–C by Lane 1D software. To normalize data collected from different gels, value at day 0 was set to one and fold changes at other time points were calculated. Data are average values from three independent experiments. Cells were grown without stress (CK, control) or in the dark, cold (5 °C), heat (40 °C), or with NaCl or glucose supplementation. Samples were collected on days 0, 1, 2, 4, and 6 of culture. Full article ">Figure 3
Recombinant protein expression and antibody verification. Candidate genes (RCK1, OEE2, FKBP12, and HSP70A-N terminal sequence) were cloned into expression vector pET30a and transformed into Escherichia coli. Expression of recombinant proteins was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to lysogeny broth (LB) medium. E. coli cells were collected by centrifugation and total proteins were extracted after sonication. Supernatant and pellet samples were separated by centrifugation. Recombinant proteins in pellet form were washed in 2 M urea and then dissolved in 8 M urea. SDS-PAGE-separated proteins were visualized by Coomassie Blue staining. Dissolved recombinant protein was used to generate polyclonal antibodies. To verify the specificity of antibodies, Western blotting analysis was carried out against dissolved proteins. M: molecular weight marker; 0 h: samples collected at 0 h; S: supernatant samples collected at 3 h after IPTG induction. P: pellet samples collected at 3 h after IPTG induction; 2 M: pellet samples washed with 2 M urea; 8 M: pellet samples dissolved in 8 M urea; WB: SDS-PAGE-separated proteins were immobilized on polyvinylidene difluoride (PVDF) membrane and detected with antibodies against recombinant proteins. Full article ">Figure 4
Western blot detection of candidate proteins in Chlamydomonas reinhardtii cells under abiotic stresses at different time points. C. reinhardtii cells cultured under different abiotic stress conditions were collected at five time points. Total proteins were extracted and separated by SDS-PAGE. PVDF-membrane-immobilized proteins were detected by antibodies against 20 candidate proteins. CK, Dark, 5 °C, 40 °C, NaCl, and Glucose are treatments for C. reinhardtii cells. D0, D1, D2, D4, and D6 are the time points (days) of sample collection. Full article ">Figure 5
Average relative fold change (ARF) of candidate proteins and selected biomarkers. ARFs calculated for candidate proteins are illustrated by different colored bars (see key). For each protein, ARF was calculated as follows: ∑1, 2, 4, 6(I(Treatment) − I(CK))/I(CK))/4, where 1, 2, 4, and 6 are days 1, 2, 4, and 6, respectively, I(treatment) is normalized intensity of Western blot for specific treatment at n (1, 2, 4, or 6) time points, and I(CK) is the normalized intensity of the Western blot for the control (CK). “*” indicates the recommended biomarker with highest ARF under multiple or specific treatments. (A) ARF value of HSP90B was highlighted with “*” indicating it is a wide-range biomarker for five treatments. HSP90B data were excluded in (B–G) and “*” designated the recommended biomarkers respectively. (B) highlights the ARF of FAP127. (C) highlights the ARF of ATPs-A. RCK1 is highlighted with “*” in (D), BCR1 in (E), MPC1 in (F) and RMT1 in (G). Full article ">

945 KiB

Open AccessReview

Principal Aspects Regarding the Maintenance of Mammalian Mitochondrial Genome Integrity

by Panagiotis V. S. Vasileiou, Iordanis Mourouzis and Constantinos Pantos

Int. J. Mol. Sci. 2017, 18(8), 1821; https://doi.org/10.3390/ijms18081821 - 22 Aug 2017

Cited by 20 | Viewed by 6443

Abstract

Mitochondria have emerged as key players regarding cellular homeostasis not only due to their contribution regarding energy production through oxidative phosphorylation, but also due to their involvement in signaling, ion regulation, and programmed cell death. Indeed, current knowledge supports the notion that mitochondrial [...] Read more.

Mitochondria have emerged as key players regarding cellular homeostasis not only due to their contribution regarding energy production through oxidative phosphorylation, but also due to their involvement in signaling, ion regulation, and programmed cell death. Indeed, current knowledge supports the notion that mitochondrial dysfunction is a hallmark in the pathogenesis of various diseases. Mitochondrial biogenesis and function require the coordinated action of two genomes: nuclear and mitochondrial. Unfortunately, both intrinsic and environmental genotoxic insults constantly threaten the integrity of nuclear as well as mitochondrial DNA. Despite the extensive research that has been made regarding nuclear genome instability, the importance of mitochondrial genome integrity has only recently begun to be elucidated. The specific architecture and repair mechanisms of mitochondrial DNA, as well as the dynamic behavior that mitochondria exert regarding fusion, fission, and autophagy participate in mitochondrial genome stability, and therefore, cell homeostasis. Full article

(This article belongs to the Special Issue Mechanisms Leading to Genomic Instability)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
The level of mitochondrial DNA damage determines whether mitophagy or cell death will be stimulated. ATM (ATM serine/threonine kinase), p53 and Sirt1 (sirtuin 1) mediate the signaling between the nucleus and the mitochondria. Full article ">Figure 2
Factors with which Sirt1 interacts regarding mitochondrial homeostasis. [PGC1a: peroxisome proliferator-activated receptor gamma co-activator (PGC1a), HIF1a: hypoxia-inducible factor 1-alpha, AMPK: 5′ adenosine monophosphate-activated protein kinase]. Full article ">Figure 3
Summary of factors mentioned in the text that are associated with instability of mammalian mitochondrial DNA. The list presented herein is not exhaustive. (Polγ: polymerase γ, mtSSB: mitochondrial single stranded DNA binding protein, TFAM: mitochondrial transcription factor A, NRF1/2: nuclear respiratory factors 1 and 2, PrimPol: primase-polymerase; Pif1: petite integration frequency 1, BER: base excision repair, Mfn: mitofusin, Opa1: optic atrophy protein 1,Drp1: dynamin related protein 1,hFIS1: fission 1 protein, Mff: mitochondrial fission factor, MiD49/51: mitochondrial dynamics proteins of 49 and 51 kDa, RRM2B: ribonucleoside-diphosphate reductase subunit M2 B). Full article ">Figure 4
Proteins involved in the maintenance of mitochondrial DNA integrity, in addition to their role in ensuring nuclear genomic homeostasis. (BRCA1: breast cancer type 1 protein, CtIP: CtBP-interacting protein, FEN1: flap endonuclease 1, MRE11: meiotic recombination 11, PARP: poly (ADP-ribose) polymerase). Full article ">

3959 KiB

Open AccessArticle

GLUT10—Lacking in Arterial Tortuosity Syndrome—Is Localized to the Endoplasmic Reticulum of Human Fibroblasts

by Alessandra Gamberucci, Paola Marcolongo, Csilla E. Németh, Nicoletta Zoppi, András Szarka, Nicola Chiarelli, Tamás Hegedűs, Marco Ritelli, Giulia Carini, Andy Willaert, Bert L. Callewaert, Paul J. Coucke, Angiolo Benedetti, Éva Margittai, Rosella Fulceri, Gábor Bánhegyi and Marina Colombi

Int. J. Mol. Sci. 2017, 18(8), 1820; https://doi.org/10.3390/ijms18081820 - 22 Aug 2017

Cited by 17 | Viewed by 5552

Abstract

GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify [...] Read more.

GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify the localization of GLUT10. In silico GLUT10 localization prediction suggested its presence in the endoplasmic reticulum (ER). Immunoblotting showed the presence of GLUT10 protein in the microsomal, but not in mitochondrial fractions of human fibroblasts and liver tissue. An even cytosolic distribution with an intense perinuclear decoration of GLUT10 was demonstrated by immunofluorescence in human fibroblasts, whilst mitochondrial markers revealed a fully different decoration pattern. GLUT10 decoration was fully absent in fibroblasts from three ATS patients. Expression of exogenous, tagged GLUT10 in fibroblasts from an ATS patient revealed a strict co-localization with the ER marker protein disulfide isomerase (PDI). The results demonstrate that GLUT10 is present in the ER. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
GLUT10 is present in the microsomal fraction of human fibroblasts and liver tissue. Subcellular fractions were prepared from human skin fibroblasts (A) or human liver tissue (B) and analyzed by Western blot after SDS-page separation of proteins, with antibodies to the GLUT10 protein as well as the marker proteins Grp94 for microsomes, VDAC1 and cyclophilin D for mitochondria, and GAPDH for the cytosol, as detailed in the “Experimental” section. Abbreviations: N: Nuclei; MT: mitochondria; Cyt: cytosol; MS: microsomes; Ho: homogenate; PNS: post nuclear supernatant. Full article ">Figure 2
GLUT10 and mitochondrial immunostaining in fibroblasts from human healthy subjects and three arterial tortuosity syndrome (ATS) patients. Fibroblasts were prepared and immunoreacted with the anti-GLUT10 antibody Ab1 and an antibody to Cyt C, and the images were acquired by fluorescent microscopy as detailed in the “Experimental” section. P1, P2 and P3 indicate three unrelated ATS patients as described in the “Experimental” section. Scale bar = 5 μm. Full article ">Figure 3
GLUT10 immunostaining and mitochondrial decoration with a fluorescent probe in the human fibroblast cell line BJ-5ta. Human fibroblast immortalized with hTERT (BJ-5ta line) were immunoreacted with the anti-GLUT10 antibody Ab2, and the mitochondrial fluorescent probe Mito Tracker™ Orange as reported in the “Experimental” section. Images were acquired by fluorescent microscopy as detailed in the “Experimental” section. Scale bar = 5 μm. Full article ">Figure 4
GLUT10 co-localizes with the ER marker protein disulfide isomerase (PDI). Tagged GLUT10 was transiently transfected in fibroblasts of the ATS patient P1 that were immunoreacted with antibodies to the GLUT10 tag and the ER marker PDI as reported in the “Experimental” section. The images were acquired by fluorescent microscopy as detailed in the “Experimental” section. Scale bar = 10 μm. Full article ">

743 KiB

Open AccessCommunication

Impact of Platelet-Rich Plasma on Viability and Proliferation in Wound Healing Processes after External Radiation

by Yvonne Reinders, Oliver Felthaus, Gero Brockhoff, Fabian Pohl, Norbert Ahrens, Lukas Prantl and Frank Haubner

Int. J. Mol. Sci. 2017, 18(8), 1819; https://doi.org/10.3390/ijms18081819 - 22 Aug 2017

Cited by 17 | Viewed by 5668

Abstract

Platelet-rich plasma is a current subject of studies on chronic wound healing therapy due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of platelet-rich plasma on irradiated cells of the cutaneous wound healing process [...] Read more.

Platelet-rich plasma is a current subject of studies on chronic wound healing therapy due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of platelet-rich plasma on irradiated cells of the cutaneous wound healing process are poorly understood so far. In this study, the interaction of endothelial cells and adipose-derived stem cells in conjunction with treatment with platelet-rich plasma is investigated in the context of radiation effects. Therefore, the expression of surface-marker CD90 and CD31 was determined. Moreover, cell proliferation and viability after external radiation was analyzed with and without treatment by platelet-rich plasma. Full article

(This article belongs to the Special Issue Molecular Research of Epidermal Stem Cells 2017)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Flow cytometric analysis of the surface markers CD90 and CD31. CD31 is highlighted in light grey, CD90 in dark grey. Samples were treated with 5% platelet-rich plasma and irradiated using a dose of 2 Gy (J/kg). Data for control cells are given. (a) Comparison of adipose-derived stem cells in α-MEM and adipose-derived stem cells in EC-Medium; (b) Analysis of CD90 and CD31 of adipose-derived stem cells, microvascular endothelial cells and the respective co-culture. Full article ">Figure 2
Cell viability determined from adipose-derived stem cells. (a) Human dermal endothelial microvascular cells; (b) and co-cultures of the respective cell lines; (c) upon external radiation and treatment with platelet-rich plasma using a WST-8 assay. Values are represented as means ± standard deviations. Statistical significances of * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 are indicated, respectively. Full article ">Figure 3
Cell proliferation of ASC, HDMEC and the corresponding co-culture upon treatment with PRP was determined using a BrdU assay. Error bars represent standard deviation. Values are represented as means ± standard deviations. Statistical significances of * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 are indicated, respectively. Full article ">

912 KiB

Open AccessCase Report

Peritoneal Mesothelioma with Residential Asbestos Exposure. Report of a Case with Long Survival (Seventeen Years) Analyzed by Cgh-Array

by Gabriella Serio, Federica Pezzuto, Andrea Marzullo, Anna Scattone, Domenica Cavone, Alessandra Punzi, Francesco Fortarezza, Mattia Gentile, Antonia Lucia Buonadonna, Mattia Barbareschi and Luigi Vimercati

Int. J. Mol. Sci. 2017, 18(8), 1818; https://doi.org/10.3390/ijms18081818 - 22 Aug 2017

Cited by 27 | Viewed by 4710

Abstract

Malignant mesothelioma is a rare and aggressive tumor with limited therapeutic options. We report a case of a malignant peritoneal mesothelioma (MPM) epithelioid type, with environmental asbestos exposure, in a 36-year-old man, with a long survival (17 years). The patient received standard treatment [...] Read more.

Malignant mesothelioma is a rare and aggressive tumor with limited therapeutic options. We report a case of a malignant peritoneal mesothelioma (MPM) epithelioid type, with environmental asbestos exposure, in a 36-year-old man, with a long survival (17 years). The patient received standard treatment which included cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Methods and Results: Molecular analysis with comparative genomic hybridization (CGH)-array was performed on paraffin-embedded tumoral samples. Multiple chromosomal imbalances were detected. The gains were prevalent. Losses at 1q21, 2q11.1→q13, 8p23.1, 9p12→p11, 9q21.33→q33.1, 9q12→q21.33, and 17p12→p11.2 are observed. Chromosome band 3p21 (BAP1), 9p21 (CDKN2A) and 22q12 (NF2) are not affected. Conclusions: the defects observed in this case are uncommon in malignant peritoneal mesothelioma. Some chromosomal aberrations that appear to be random here, might actually be relevant events explaining the response to therapy, the long survival and, finally, may be considered useful prognostic factors in peritoneal malignant mesothelioma (PMM). Full article

(This article belongs to the Special Issue Mesothelioma Heterogeneity: Potential Mechanisms)

► Show Figures

Figure 1

338 KiB

Open AccessReview

Current Advances in Thyroid Cancer Management. Are We Ready for the Epidemic Rise of Diagnoses?

by Dagmara Rusinek, Ewa Chmielik, Jolanta Krajewska, Michal Jarzab, Malgorzata Oczko-Wojciechowska, Agnieszka Czarniecka and Barbara Jarzab

Int. J. Mol. Sci. 2017, 18(8), 1817; https://doi.org/10.3390/ijms18081817 - 22 Aug 2017

Cited by 35 | Viewed by 7041

Abstract

A rising incidence of thyroid cancers (TCs) mainly small tumors, observed during recent years, lead to many controversies regarding treatment strategies. TCs represent a distinct molecular background and clinical outcome. Although in most cases TCs are characterized by a good prognosis, there are [...] Read more.

A rising incidence of thyroid cancers (TCs) mainly small tumors, observed during recent years, lead to many controversies regarding treatment strategies. TCs represent a distinct molecular background and clinical outcome. Although in most cases TCs are characterized by a good prognosis, there are some aggressive forms, which do not respond to standard treatment. There are still some questions, which have to be resolved to avoid dangerous simplifications in the clinical management. In this article, we focused on the current advantages in preoperative molecular diagnostic tests and histopathological examination including noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). We discussed the controversies regarding the extent of thyroid surgery and adjuvant radioiodine therapy, as well as new treatment modalities for radioiodine-refractory differentiated thyroid cancer (RR-DTC). Considering medullary thyroid cancer (MTC), we analyzed a clinical management based on histopathology and RET (ret proto-oncogene) mutation genotype, disease follow-up with a special attention to serum calcitonin doubling time as an important prognostic marker, and targeted therapy applied in advanced MTC. In addition, we provided some data regarding anaplastic thyroid cancer (ATC), a highly lethal neoplasm, which lead to death in nearly 100% of patients due to the lack of effective treatment options. Full article

(This article belongs to the Special Issue Cell and Molecular Biology of Thyroid Disorders)

► Show Figures

Graphical abstract

1292 KiB

Open AccessArticle

Bioaccessibility, Intestinal Permeability and Plasma Stability of Isorhamnetin Glycosides from Opuntia ficus-indica (L.)

by Marilena Antunes-Ricardo, César Rodríguez-Rodríguez, Janet A. Gutiérrez-Uribe, Eduardo Cepeda-Cañedo and Sergio O. Serna-Saldívar

Int. J. Mol. Sci. 2017, 18(8), 1816; https://doi.org/10.3390/ijms18081816 - 22 Aug 2017

Cited by 41 | Viewed by 5745

Abstract

Isorhamnetin glycosides are representative compounds of Opuntia ficus-indica that possess different biological activities. There is slight information about the changes in bioaccessibility induced by the glycosylation pattern of flavonoids, particularly for isorhamnetin. In this study, the bioaccessibility and permeability of isorhamnetin glycosides extracted [...] Read more.

Isorhamnetin glycosides are representative compounds of Opuntia ficus-indica that possess different biological activities. There is slight information about the changes in bioaccessibility induced by the glycosylation pattern of flavonoids, particularly for isorhamnetin. In this study, the bioaccessibility and permeability of isorhamnetin glycosides extracted from O. ficus-indica were contrasted with an isorhamnetin standard. Also, the plasma stability of these isorhamnetin glycosides after intravenous administration in rats was evaluated. Recoveries of isorhamnetin after oral and gastric digestion were lower than that observed for its glycosides. After intestinal digestion, isorhamnetin glycosides recoveries were reduced to less than 81.0%. The apparent permeability coefficient from apical (AP) to basolateral (BL) direction (Papp_(AP-BL)) of isorhamnetin was 2.6 to 4.6-fold higher than those obtained for its glycosides. Isorhamnetin diglycosides showed higher Papp_(AP-BL) values than triglycosides. Sugar substituents affected the Papp_(AP-BL) of the triglycosides. Isorhamnetin glycosides were better retained in the circulatory system than the aglycone. After intravenous dose of the isorhamnetin standard, the elimination half-life was 0.64 h but increased to 1.08 h when the O. ficus-indica extract was administered. These results suggest that isorhamnetin glycosides naturally found in O. ficus-indica could be a controlled delivery system to maintain a constant plasmatic concentration of this important flavonoid to exert its biological effects in vivo. Full article

(This article belongs to the Special Issue Bioactive Phenolics and Polyphenols 2018)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Chemical structures of isorhamnetin glycosides found in O. ficus-indica extract. Glc = glucose; Rha = rhamnose; Pen = pentose. Full article ">Figure 2
Comparison between permeability of isorhamnetin glycosides from O. ficus-indica extract and isorhamnetin standard in a Caco-2/HT-29 cell monolayer. (a) Apparent permeability (Papp) of isorhamnetin glycosides (IGRR, IGRP, IGP, IGR) and isorhamnetin standard from apical to basolateral (AP-BL), and basolateral to apical compartment (BL-AP); (b) efflux ratio of isorhamnetin glycosides (IGRR, IGRP, IGP, IGR) and isorhamnetin standard. Data was expressed as mean ± standard deviation. IGRR: isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside; IGRP: isorhamnetin-3-O-glucosyl-rhamnosyl-pentoside; IGP: isorhamnetin-3-O-glucosyl-pentoside; IGR: isorhamnetin-3-O-glucosyl-rhamnoside; I: isorhamnetin. Different letters indicate significant differences among different isorhamnetin glycosides. Efflux ratio was calculated by the relation between Papp(BL-AP)/Papp(AP-BL). Full article ">Figure 3
Chromatograms showing isorhamnetin release from isorhamnetin glycosides contained in O. ficus-indica extract, using (a) 20 min; (b) 90 min; or (c) 180 min of acid hydrolysis; (d) isorhamnetin recovery percentages obtained after 20, 90, or 180 min of acid hydrolysis. Different letters indicate significant differences among different isorhamnetin glycosides. Full article ">Figure 4
Isorhamnetin concentration (µmol·L−1) in rat plasma and pharmacokinetic parameters after an intravenous dose (2 mg/kg) of an O. ficus-indica extract or isorhamnetin standard. Full article ">

6958 KiB

Open AccessArticle

Successive Onset of Molecular, Cellular and Tissue-Specific Responses in Midgut Gland of Littorina littorea Exposed to Sub-Lethal Cadmium Concentrations

by Denis Benito, Michael Niederwanger, Urtzi Izagirre, Reinhard Dallinger and Manu Soto

Int. J. Mol. Sci. 2017, 18(8), 1815; https://doi.org/10.3390/ijms18081815 - 22 Aug 2017

Cited by 24 | Viewed by 5560

Abstract

Cadmium (Cd) is one of the most harmful metals, being toxic to most animal species, including marine invertebrates. Among marine gastropods, the periwinkle (Littorina littorea) in particular can accumulate high amounts of Cd in its midgut gland. In this organ, the [...] Read more.

Cadmium (Cd) is one of the most harmful metals, being toxic to most animal species, including marine invertebrates. Among marine gastropods, the periwinkle (Littorina littorea) in particular can accumulate high amounts of Cd in its midgut gland. In this organ, the metal can elicit extensive cytological and tissue-specific alterations that may reach, depending on the intensity of Cd exposure, from reversible lesions to pathological cellular disruptions. At the same time, Littorina littorea expresses a Cd-specific metallothionein (MT) that, due to its molecular features, expectedly exerts a protective function against the adverse intracellular effects of this metal. The aim of the present study was, therefore, to assess the time course of MT induction in the periwinkle’s midgut gland on the one hand, and cellular and tissue-specific alterations in the digestive organ complex (midgut gland and digestive tract) on the other, upon exposure to sub-lethal Cd concentrations (0.25 and 1 mg Cd/L) over 21 days. Depending on the Cd concentrations applied, the beginning of alterations of the assessed parameters followed distinct concentration-dependent and time-dependent patterns, where the timeframe for the onset of the different response reactions became narrower at higher Cd concentrations compared to lower exposure concentrations. Full article

(This article belongs to the Special Issue Metal Metabolism in Animals II)

► Show Figures

Graphical abstract

2809 KiB

Open AccessArticle

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo

by Ute Wölfle, Birgit Haarhaus, Jasmin Seiwerth, Anja Cawelius, Kay Schwabe, Karl-Werner Quirin and Christoph M. Schempp

Int. J. Mol. Sci. 2017, 18(8), 1814; https://doi.org/10.3390/ijms18081814 - 22 Aug 2017

Cited by 31 | Viewed by 7208

Abstract

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, [...] Read more.

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema. Full article

(This article belongs to the Special Issue Inflammatory Skin Conditions 2017)

► Show Figures

Graphical abstract

1063 KiB

Open AccessReview

Towards a Better Understanding of GABAergic Remodeling in Alzheimer’s Disease

by Karan Govindpani, Beatriz Calvo-Flores Guzmán, Chitra Vinnakota, Henry J. Waldvogel, Richard L. Faull and Andrea Kwakowsky

Int. J. Mol. Sci. 2017, 18(8), 1813; https://doi.org/10.3390/ijms18081813 - 21 Aug 2017

Cited by 143 | Viewed by 13142

Abstract

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the vertebrate brain. In the past, there has been a major research drive focused on the dysfunction of the glutamatergic and cholinergic neurotransmitter systems in Alzheimer’s disease (AD). However, there is now growing evidence [...] Read more.

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the vertebrate brain. In the past, there has been a major research drive focused on the dysfunction of the glutamatergic and cholinergic neurotransmitter systems in Alzheimer’s disease (AD). However, there is now growing evidence in support of a GABAergic contribution to the pathogenesis of this neurodegenerative disease. Previous studies paint a complex, convoluted and often inconsistent picture of AD-associated GABAergic remodeling. Given the importance of the GABAergic system in neuronal function and homeostasis, in the maintenance of the excitatory/inhibitory balance, and in the processes of learning and memory, such changes in GABAergic function could be an important factor in both early and later stages of AD pathogenesis. Given the limited scope of currently available therapies in modifying the course of the disease, a better understanding of GABAergic remodeling in AD could open up innovative and novel therapeutic opportunities. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

► Show Figures

Figure 1

1706 KiB

Open AccessReview

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors

by Erzsébet Bartolák-Suki, Jasmin Imsirovic, Yuichiro Nishibori, Ramaswamy Krishnan and Béla Suki

Int. J. Mol. Sci. 2017, 18(8), 1812; https://doi.org/10.3390/ijms18081812 - 21 Aug 2017

Cited by 120 | Viewed by 15289

Abstract

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided [...] Read more.

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging. Full article

(This article belongs to the Special Issue Mitochondria Crosstalks with other Organelles in Pathophysiology)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
The mitochondria are the powerhouses of the cell. Left: A cartoon of a mitochondrion showing its outer and inner membranes, the cristae, and the matrix. Fat and sugar enter the mitochondria through channels of the outer membrane. Right: The Krebs, or citric acid, cycle feeds the chain of respiratory complexes I through IV which create an electrical and proton (H+) gradient, the electromotive force across the inner membrane. ATP synthase utilizes the electromotive force to generate ATP from ADP and inorganic phosphate (Pi) (Right image from Wikipedia [<a href="#B24-ijms-18-01812" class="html-bibr">24</a>]). Full article ">Figure 2
Intracellular and extracellular processes contributing to mitochondrial structure and function. Intracellular mitochondrial (orange) dynamics include the processes of fusion, fission, mitophagy, and biogenesis. See text for explanation for how the various molecules such as OPA1, Mfn1, Mfn2, and DRP1 govern these processes. The grey mitochondrion is damaged and degraded by mitophagy. Green lines represent microtubules, along which small mitochondrial clusters can travel with the aid of motor proteins. The dashed line represents the site of fission and the red arrows indicate the cyclic nature of fission and fusion. Note that biogenesis increases mitochondrial volume and is regulated by the peroxisome proliferator-activated receptor γ coactivator (PGC-1α). Cells are connected to the ECM (extracellular matrix) and exposed to external mechanical forces (F) at focal adhesions (FA), involving integrin receptors (Int) on the cell surface and Arg-Gly-Asp (RGD) binding sites on collagens fibers (Col-I). The cytoskeleton is linked to FAs and therefore mechanical forces from the ECM are transmitted to the mitochondria. Full article ">Figure 3
Cells were cultured on elastic membranes that could be stretched equibiaxially in a stretching device. (A) A cell labeled for cytosol (green), mitochondria (red: tetramethylrhodamine methyl ester, TMRM), and nucleus (blue) at 0% (left) and 14% (right) strains. (B) The top row shows the mitochondrial network of an entire cell imaged during constant strain application at increments of 0, 7, 14, and 30% change in membrane surface area. The bottom row shows the zoomed-in details of an individual cluster (green rectangle in top row) changing shape as higher strains are applied (green arrow), as well as a cluster undergoing fission and splitting into two smaller clusters (red arrow) [<a href="#B77-ijms-18-01812" class="html-bibr">77</a>]. Full article ">Figure 4
Relationship between complexity, measured by the fractal dimension Df, and function, assessed by a fluorescent dye (TMRM, see text) intensity that is related to ATP production rate in VSMCs. There is a linear relation between the log of ATP production and Df. The dots represent binned data from about 2000 cells showing unstretched control cells (US), 4 h of monotonously stretched (MS) cells (10% area strain at 1 Hz), and 4 h of stretching cells with a variable stretch (VS) pattern in which every cycle is different with the amplitudes uniformly distributed between 7.5% and 12.5% area strain. The US cells are in the lower left corner. These cells produce little energy and their mitochondrial fractal organization is the least complex. MS cells produce somewhat more energy and their Df is also higher, whereas VS cells produce the most ATP and have the highest complexity in terms of their fractal organization. The images show mitochondrial networks corresponding to US, MS, and VS cells. ATP production rate is related to the intensity of red color. The results were obtained by reanalyzing the data from [<a href="#B14-ijms-18-01812" class="html-bibr">14</a>]. Full article ">Figure 5
Mitochondrial structure-function relations as a function of substrate stiffness. VSMCs were seeded on substrates of different stiffness, labeled with TMRM, and cluster sizes and mean intensities were measured. The horizontal lines in the boxes are the median, the box represents the 25th percentile, and the horizontal bars are the 75th percentile of the data. The symbols are data outside the 75th percentile. (A) Cluster sizes were stiffness dependent, but only the clusters on 12.5 kPa stiffness were different from the rest. (B) Intensities were also stiffness dependent, and again only data on 12.5 kPa stiffness were different from the rest. Full article ">

1673 KiB

Open AccessArticle

NPFFR2 Activates the HPA Axis and Induces Anxiogenic Effects in Rodents

by Ya-Tin Lin, Yu-Lian Yu, Wei-Chen Hong, Ting-Shiuan Yeh, Ting-Chun Chen and Jin-Chung Chen

Int. J. Mol. Sci. 2017, 18(8), 1810; https://doi.org/10.3390/ijms18081810 - 21 Aug 2017

Cited by 23 | Viewed by 11769

Abstract

Neuropeptide FF (NPFF) belongs to the RFamide family and is known as a morphine-modulating peptide. NPFF regulates various hypothalamic functions through two receptors, NPFFR1 and NPFFR2. The hypothalamic-pituitary-adrenal (HPA) axis participates in physiological stress response by increasing circulating glucocorticoid levels and modulating emotional [...] Read more.

Neuropeptide FF (NPFF) belongs to the RFamide family and is known as a morphine-modulating peptide. NPFF regulates various hypothalamic functions through two receptors, NPFFR1 and NPFFR2. The hypothalamic-pituitary-adrenal (HPA) axis participates in physiological stress response by increasing circulating glucocorticoid levels and modulating emotional responses. Other RFamide peptides, including neuropeptide AF, neuropeptide SF and RFamide related peptide also target NPFFR1 or NPFFR2, and have been reported to activate the HPA axis and induce anxiety- or depression-like behaviors. However, little is known about the action of NPFF on HPA axis activity and anxiety-like behaviors, and the role of the individual receptors remains unclear. In this study, NPFFR2 agonists were used to examine the role of NPFFR2 in activating the HPA axis in rodents. Administration of NPFFR2 agonists, dNPA (intracerebroventricular, ICV) and AC-263093 (intraperitoneal, IP), time-dependently (in rats) and dose-dependently (in mice) increased serum corticosteroid levels and the effects were counteracted by the NPFF receptor antagonist, RF9 (ICV), as well as corticotropin-releasing factor (CRF) antagonist, α-helical CRF(9-41) (intravenous, IV). Treatment with NPFFR2 agonist (AC-263093, IP) increased c-Fos protein expression in the hypothalamic paraventricular nucleus and induced an anxiogenic effect, which was evaluated in mice using an elevated plus maze. These findings reveal, for the first time, that the direct action of hypothalamic NPFFR2 stimulates the HPA axis and triggers anxiety-like behaviors. Full article

(This article belongs to the Special Issue Role of the Hypothalamo–Pituitary–Adrenal (HPA) Axis in Health and Disease)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Effect of AC-263093 on the hypothalamic-pituitary-adrenal (HPA) axis in mice. Mice were intraperitoneal (IP) injected with vehicle or AC-263093 (5, 7.5, 10, 20, 30 mg/kg) and sacrificed after 1 h. Levels of serum corticosteroid (CORT) were measured by ELISA. Data are expressed as mean ± standard error of the mean (SEM) and were analyzed using a one-way ANOVA followed by Newman-Keuls post hoc tests. *, p < 0.05; ***, p < 0.001, compared to vehicle controls (n = 5 per group). Full article ">Figure 2
Time-dependent effects of treatment with dNPA on serum CORT in rats. Rats were injected with vehicle or dNPA (10 nmol, intracerebroventricular, ICV) and serum CORT was monitored up to 70 min following injection. (A) Levels of serum CORT were measured to indicate the activity of the HPA axis. Neuropeptide FF (NPFF) receptor antagonist RF9 (10 nmol, ICV) was administered 15 min prior to dNPA treatment. Data are expressed as mean ± SEM and were analyzed using a two-way ANOVA following by Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls. # p < 0.05; ## p < 0.01, compared dNPA to RF9+dNPA group; (B) The results of area under curve (AUC) calculation. Data are expressed as mean ± SEM and were analyzed using a one-way ANOVA following by Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls (n = 4–7 per group). Full article ">Figure 3
Time-dependent effects of treatment with AC-263093 on serum CORT in rats. Rats were injected with vehicle or AC-263093 (30 mg/kg, IP) and serum CORT was monitored for up to 90 min post-administration. (A) Levels of serum CORT were measured by ELISA. Corticotropin-releasing factor (CRF) antagonist α-helical CRF(9-41) (200 μg, intravenous, IV) was applied 15 min prior to AC-263093 treatment. Data are expressed as mean ± SEM and analyzed using a two-way ANOVA following by Bonferroni post hoc tests. * p < 0.05, compared AC-263093 to vehicle controls; (B) The results of AUC calculation. Data are expressed as mean ± SEM and were analyzed using a one-way ANOVA following by Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls (n = 4–11 per group). Full article ">Figure 4
Effects of AC-263093 treatment on c-Fos expression in paraventricular nucleus (PVN) neurons. Mice were injected with vehicle or AC-263093 (30 mg/kg, IP) and sacrificed after 1 h. (A) Immunofluorescence staining was used to detect c-Fos expression in the PVN. Nuclei were counterstained with DAPI; (B) Quantification of c-Fos-positive cell numbers in the PVN. Histogram B shows the c-Fos immunoreactive neurons counts expressed as sum of bilateral frontal cross section surface areas of PVN. Data are expressed as mean ± SEM and were analyzed using an unpaired Student’s t-test. **, p < 0.01, compared to vehicle control (n = 4 per group). Scale bar = 100 μm. Full article ">Figure 5
Effects of AC-263093 treatment on anxiety-like behavior. Mice were injected with AC-263093 (30 mg/kg, IP) 1 h prior to behavioral testing. Anxiety-like behavior was evaluated by the elevated plus maze (EPM). (A) Time that mice stayed in the open arms or closed arms; (B) Number of entries into open arms; (C) Total time spent with movement. Data are expressed as mean ± SEM and were analyzed using an unpaired Student’s t-test. *, p < 0.05; **, p < 0.01, compared to vehicle controls (n = 7–8 per group). Full article ">

2020 KiB

Open AccessArticle

Protocatechuic Acid from Pear Inhibits Melanogenesis in Melanoma Cells

by Xuan T. Truong, Seo-Hee Park, Yu-Geon Lee, Hang Yeon Jeong, Jae-Hak Moon and Tae-Il Jeon

Int. J. Mol. Sci. 2017, 18(8), 1809; https://doi.org/10.3390/ijms18081809 - 21 Aug 2017

Cited by 36 | Viewed by 12020

Abstract

Despite the critical role of melanin in the protection of skin against UV radiation, excess production of melanin can lead to hyperpigmentation and skin cancer. Pear fruits are often used in traditional medicine for the treatment of melasma; therefore, we investigated the effects [...] Read more.

Despite the critical role of melanin in the protection of skin against UV radiation, excess production of melanin can lead to hyperpigmentation and skin cancer. Pear fruits are often used in traditional medicine for the treatment of melasma; therefore, we investigated the effects of pear extract (PE) and its component, protocatechuic acid (PCA), on melanogenesis in mouse melanoma cells. We found that PE and PCA significantly suppressed melanin content and cellular tyrosinase activity through a decrease in the expression of melanogenic enzymes and microphthalmia-associated transcription factor (Mitf) in α-melanocyte stimulating hormone-stimulated mouse melanoma cells. Moreover, PCA decreased cyclic adenosine monophosphate (cAMP) levels and cAMP-responsive element-binding protein phosphorylation, which downregulated Mitf promoter activation and subsequently mediated the inhibition of melanogenesis. These results suggested that pear may be an effective skin lightening agent that targets either a tyrosinase activity or a melanogenic pathway. Full article

(This article belongs to the Special Issue Melanins and Melanogenesis: From Nature to Applications)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Effects of pear extract (PE) on melanogenesis in mouse melanoma cells. (a) B16F10 cells were treated with various concentrations of PE for 72 h. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. * p < 0.05 vs. control dimethyl sulfoxide (DMSO); (b) Cells were exposed to 1 μM α-melanocyte stimulating hormone (α-MSH) in presence or absence of PE (mg/mL) or 2 mM arbutin (Ar) for 72 h. Cell pellet images were taken using a digital camera and melanin contents were measured with synthetic melanin as standard. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; ## p < 0.01 vs. α-MSH; (c) Mushroom tyrosinase activity in the absence or presence of PE (0.5 mg/mL) or arbutin (2 mM). * p < 0.05 vs. DMSO; ** p < 0.01 vs. DMSO. Full article ">Figure 2
Effects of PE on expression of melanogenic genes. B16F10 cells were exposed to α-MSH in presence or absence of PE (mg/mL) for 24 h. (a) mRNA levels for Mitf-M, Tyr, Trp1, and Trp2 were analyzed by RT-qPCR. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; ## p < 0.01 vs. α-MSH; ### p < 0.001 vs. α-MSH. Data are mean ± SEM; n = 3; (b) Cell lysates were analyzed for microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) protein levels by immunoblotting. Full article ">Figure 3
Effects of protocatechuic acid (PCA) on melanin synthesis and tyrosinase activity. (a) Structure of PCA; (b) B16F10 cells were treated with various concentrations of PCA for 72 h. The cell viability was measured by MTT assay; (c) Cells were exposed to 1 μM α-MSH in presence or absence of PCA (μM) for 72 h. Melanin contents were measured with synthetic melanin as standard. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; (d) Cells were exposed to 1 μM α-MSH in presence or absence of PCA (μM) for 24 h. Cellular tyrosinase activity. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; (e) Mushroom tyrosinase activity in a cell free system. Data are mean ± SEM; n = 3. Full article ">Figure 4
Effects of PCA on expression of melanogenic genes and activation of MITF promoter. B16F10 cells were exposed to α-MSH in presence or absence of PCA (μM) for 24 h. (a) mRNA levels for Mitf-M, Tyr, Trp1, and Trp2 were analyzed by RT-qPCR. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH. Data are mean ± SEM; n = 3; (b) Cell lysates were analyzed for MITF, TYR, TRP1, and TRP2 protein levels by immunoblotting; (c) Reporter assays were performed using mouse MITF-M promoter construct. Cells were transfected with wild type (wt) cAMP-responsive element (CRE) or mtCRE luciferase reporters for 24 h before treatment with PCA (μM) and/or α-MSH for 24 h. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; ## p < 0.01 vs. α-MSH; ### p < 0.005 vs. α-MSH. Data are mean ± SEM; n = 3. Full article ">Figure 5
Effects of PCA on CREB phosphorylation and intracellular cAMP level. B16F10 cells were pretreated with 100 μM PCA for 24 h prior to 1 μM α-MSH treatment for indicated time. (a) Cell lysates were analyzed for pCREB, CREB, and GAPDH protein levels by immunoblotting; (b) mRNA levels for Pgc-1α were analyzed by RT-qPCR. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; (c) Cells were pretreated with 100 μM PCA for 24 h, and then exposed to 1 μM α-MSH for 30 min. Intracellular cAMP concentration was measured by immunoassay as described in Materials and methods. * p < 0.05 vs. control; # p < 0.05 vs. α-MSH; (d) Cells were exposed to 20 μM forskolin (Fsk) or 100 μM 8-Br-cAMP in presence or absence of PCA (100 μM) for 24 h. mRNA levels for Mitf-M and Pgc-1α. # p < 0.05 vs. Fsk; ns, not significant. Data are mean ± SEM; n = 3; (e) Protein level for MITF. Full article ">Figure 6
A proposed mechanism for inhibitory effect of PE and its components (PCA and arbutin) on melanogenesis. Arrows indicate positive regulation while T-bars denote inhibitory effects. Full article ">

6318 KiB

Open AccessArticle

Muscle Conditional Medium Reduces Intramuscular Adipocyte Differentiation and Lipid Accumulation through Regulating Insulin Signaling

by Haiyin Han, Wei Wei, Weiwei Chu, Kaiqing Liu, Ye Tian, Zaohang Jiang and Jie Chen

Int. J. Mol. Sci. 2017, 18(8), 1799; https://doi.org/10.3390/ijms18081799 - 20 Aug 2017

Cited by 14 | Viewed by 6375

Abstract

Due to the paracrine effects of skeletal muscle, the lipid metabolism of porcine intramuscular (i.m.) preadipocytes was different from that of subcutaneous (s.c.) preadipocytes. To investigate the development of i.m. preadipocytes in vivo, the s.c. preadipocytes were cultured with muscle conditional cultured medium [...] Read more.

Due to the paracrine effects of skeletal muscle, the lipid metabolism of porcine intramuscular (i.m.) preadipocytes was different from that of subcutaneous (s.c.) preadipocytes. To investigate the development of i.m. preadipocytes in vivo, the s.c. preadipocytes were cultured with muscle conditional cultured medium (MCM) for approximating extracellular micro-environment of the i.m. preadipocytes. Insulin signaling plays a fundamental role in porcine adipocyte differentiation. The expression levels of insulin receptor (INSR) and insulin-like growth factor 1 receptor (IGF-1R) in i.m. Preadipocytes were higher than that in s.c. preadipocytes. The effects of MCM on adipocyte differentiation, lipid metabolism and insulin signaling transdution were verified. MCM induced the apoptosis of s.c. preadipocytes but not of s.c. adipocytes. Moreover, MCM inhibited adipocyte differentiation at pre-differentiation and early stages of differentiation, while the expression levels of INSR and IGF-1R were increased. Furthermore, MCM treatment increased adipocyte lipolysis and fatty acid oxidation through induction of genes involved in lipolysis, thermogenesis, and fatty acid oxidation in mitochondria. Consistent with the above, treatment of s.c. adipocytes with MCM upregulated mitochondrial biogenesis. Taken together, MCM can approximate the muscle micro-environment and reduce intramuscular adipocyte differentiation and lipid accumulation via regulating insulin signaling. Full article

(This article belongs to the Special Issue Adipose Stem Cells)

► Show Figures

Figure 1

Figure 1
Identification of porcine i.m. and s.c. preadipocytes. The dedifferentiated porcine intramuscular (i.m.) and subcutaneous (s.c.) preadipocytes showed fibroblast-like morphology (A); The i.m. and s.c. preadipocytes were induced in adipogenic medium at confluence. Nine days later, the i.m. and s.c. preadipocytes were re-differentiated to mature adipocytes with lipid droplets (B); The isolated i.m. and s.c. preadipocytes were seeded in 12-well plates, and the cells of day 3 were detected by Pref-1 immunofluorescent staining (C). Scale bar = 100 μm. Full article ">Figure 2
Downregulation of insulin receptor (INSR) or IGF-1 receptor (IGF-1R) by siRNA interference decreased porcine adipocyte differentiation. The siRNAs targeting INSR and IGF-1R (si-INSR, si-IGF-1R) were transfected into porcine s.c. preadipocytes. The INSR/IGF-1R mRNA (A) (n = 6) and protein levels (B) (n = 3) were verified after transfected with siNC and si-INSR/si-IGF-1R at 24 and 48 h, respectively; Transfected s.c. preadipocytes were induced to differentiate for nine days; The intracellular accumulation was determined by cytoplasm triglyceride content (C) (n = 4) and Oil Red O staining (D) (n = 3). The mRNA expression differences were normalized to Ribosomal protein lateral stalk subunit P0 (RPLP0) mRNA level. The β-actin bands served as an internal control for protein loading. ** p < 0.01, Scale bar = 200 μm. Full article ">Figure 3
INSR and IGF-1R expression pattern in i.m. and s.c. preadipocytes. The comparison of INSR and IGF-1R mRNA expression levels in i.m. and s.c. preadipocytes (A) (n = 3); The protein expression level of IGF-1R in i.m. and s.c. preadipocytes was detected by Western blot and subsequently quantified. (B) (n = 3). The mRNA expression differences were normalized to RPLP0 mRNA level. The β-actin bands served as an internal control for protein loading. ** p < 0.01. Full article ">Figure 4
The inhibitory effect of muscle conditional medium (MCM) on s.c. preadipocytes differentiation. Confluenced s.c. preadipocytes were incubated in adipogenic medium supplemented with an increasing amount of MCM at the indicated total protein concentration (n = 3). After nine days, adipocytes were staining with Oil Red O. Scale bar = 200 μm. Full article ">Figure 5
Flow cytometric detection and quantification of MCM-induced apoptosis in porcine s.c. preadipocytes and s.c. adipocytes. Porcine s.c. preadipocytes (A) and s.c. adipocytes (B) were cultured for three days with MCM versus physiological saline, and stained with fluorescein isothiocyanate (FITC)-labled annexin V (Annexin V-FITC) and propidium iodine (PI); then, the cell apoptosis was analyzed. n = 3, * p < 0.05, ** p < 0.01, Scale bar = 200 μm. Full article ">Figure 6
The effective duration of activity of MCM during adipocyte differentiation. s.c. preadipocytes were treated for three days with MCM at 120 μg mL−1 total protein concentration for four different time intervals: pre-differentiation (day –3 to 0), early (day 0 to 3), middle (day 4 to 6), and late (day 7 to 9) stages of differentiation. After nine days of differentiation, the effects of MCM on intracellular oil droplets (A) (n = 3), triglycerides content (B) (n = 3) and peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid binding protein (FABP4) mRNA expression (C) (n = 6) were verified. The mRNA expression differences were normalized to RPLP0 mRNA levels. Triglyceride content was normalized to protein content. * p < 0.05, ** p < 0.01, Scale bar = 200 μm. Full article ">Figure 7
The genes expression patterns in the INSR/IGF-1R-IRS-1 signaling. The mRNA expression of INSR, IGF-1R and IRS-1 with MCM treatment at the pre-differentiation stage of adipocyte differentiation for three days (A) (n = 4); The protein abundance of IGF-1R, IRS-1, and p-IRS-1 with MCM treatment at pre-differentiation stage for three days (B) and protein quantified was shown (C) (n = 3 for control, n = 4 for treatment); The mRNA expression of INSR (D) (n = 6), IGF-1R ((E) n = 6), and IRS-1 ((F) n = 6) with MCM treatment for different durations; The protein abundance of IGF-1R, IRS-1, and p-IRS-1 with MCM treatment during different differentiation durations (G) and protein quantified was shown ((H) n = 3); The mRNA expression differences were normalized to RPLP0 mRNA level. The β-actin bands served as an internal control for protein loading, p-IRS-1 (Try) expression was normalized to IRS-1 expression. * p < 0.05, ** p < 0.01. Full article ">Figure 8
Effect of MCM on adipocyte lipolysis and fatty acid oxidation. Fully differentiated adipocytes were incubated with the Dulbecco’s Modified Eagle’s medium (DMEM) (no phenol red) with MCM for 24 h to verify the lipolytic effects of MCM. The glycerol levels in the culture medium were assayed (A); Adipocytes were cultured with MCM for three days. The expression levels of genes involved in lipolysis (ATGL and HSL) and fatty acid synthesis (FASN and ACC) (B) were detected by q PCR, respectively; Relative mRNA expression of genes responsible for fatty acid oxidation (PPARα, Cpt-1α, and Cpt-1β) (C) and thermogenesis (PGC-1α, PRDM16, and Cidea) (D) was detected; Relative abundance of mitochondrial DNA (mtDNA) (E) and the genes responsible for mitochondrial biogenesis (PGC-1α, TRFM, and NRF) (F) were detected. Dates were normalized to the mRNA levels of RPLP0. n = 6, * p < 0.05, ** p < 0.01. Full article ">

891 KiB

Open AccessReview

Crosstalk between DNA Damage and Inflammation in the Multiple Steps of Carcinogenesis

by Shosuke Kawanishi, Shiho Ohnishi, Ning Ma, Yusuke Hiraku and Mariko Murata

Int. J. Mol. Sci. 2017, 18(8), 1808; https://doi.org/10.3390/ijms18081808 - 19 Aug 2017

Cited by 188 | Viewed by 11722

Abstract

Inflammation can be induced by chronic infection, inflammatory diseases and physicochemical factors. Chronic inflammation is estimated to contribute to approximately 25% of human cancers. Under inflammatory conditions, inflammatory and epithelial cells release reactive oxygen (ROS) and nitrogen species (RNS), which are capable of [...] Read more.

Inflammation can be induced by chronic infection, inflammatory diseases and physicochemical factors. Chronic inflammation is estimated to contribute to approximately 25% of human cancers. Under inflammatory conditions, inflammatory and epithelial cells release reactive oxygen (ROS) and nitrogen species (RNS), which are capable of causing DNA damage, including the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-nitroguanine. We reported that 8-nitroguanine was clearly formed at the sites of cancer induced by infectious agents including Helicobacter pylori, inflammatory diseases including Barrett’s esophagus, and physicochemical factors including asbestos. DNA damage can lead to mutations and genomic instability if not properly repaired. Moreover, DNA damage response can also induce high mobility group box 1-generating inflammatory microenvironment, which is characterized by hypoxia. Hypoxia induces hypoxia-inducible factor and inducible nitric oxide synthase (iNOS), which increases the levels of intracellular RNS and ROS, resulting DNA damage in progression with poor prognosis. Furthermore, tumor-producing inflammation can induce nuclear factor-κB, resulting in iNOS-dependent DNA damage. Therefore, crosstalk between DNA damage and inflammation may play important roles in cancer development. A proposed mechanism for the crosstalk may explain why aspirin decreases the long-term risk of cancer mortality. Full article

(This article belongs to the Special Issue Mechanisms Leading to Genomic Instability)

► Show Figures

Figure 1

Figure 1
Mechanism for DNA damage in epithelial cells by inflammation. NO and O2− are produced during inflammation. Although NO is sufficiently long-lived to diffuse through the extracellular matrix, and enter the nucleus, O2− released by neutrophils or macrophages during inflammation is not sufficiently long-lived. Alternatively, inflammatory cells may use cytokines such as TNF-α to stimulate O2− formation via Nox in neighboring epithelial cells. NO, which is generated by especially iNOS, reacts with O2− forming ONOO−, which causes mutagenic DNA damage, such as 8-nitroguanine and 8-oxodG. NO and ROS can participate in inhibition of a number of DNA repair enzymes, which enhances mutations, leading to genomic instability. Full article ">Figure 2
Mechanism for crosstalk between DNA damage and inflammation in the multiple steps of carcinogenesis. In cancer cells, cell death, which is induced by ROS and RNS, can induce HMGB1 expression. HMGB1 is passively released into the extracellular space from damaged or necrotic cells. Hypoxia induces HIF-1 expression, which regulates iNOS expression, and promotes translocation of HMGB1 from the nucleus to the cytoplasm. Via TLR4, extracellular HMGB1 can promote NF-κB transportation to the nucleus and induce the expression of TNF-α and IL-6, which induce iNOS expression. NO, which is generated by iNOS in tumor-associated macrophage (TAM), is released to the extracellular space and interacts with O2− generated via Nox in cancer cells to form ONOO−. ONOO− causes DNA damage resulting in mutations and genomic instability as not properly repaired. NF-κB induces not only iNOS but also COX-2 expression. COX-2 participates in formation of PGE2, which induces iNOS. Aspirin can inhibit COX-2, resulting in the decrease of PGE2 and iNOS and suppression of the crosstalk between DNA damage and inflammation in cancer development. This is a possible mechanism by which aspirin reduces the long-term risk of death due to cancer. Full article ">

3009 KiB

Open AccessArticle

Signaling Cascade Involved in Rapid Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Dexamethasone

by Miriam Bossmann, Benjamin W. Ackermann, Ulrich H. Thome and Mandy Laube

Int. J. Mol. Sci. 2017, 18(8), 1807; https://doi.org/10.3390/ijms18081807 - 19 Aug 2017

Cited by 6 | Viewed by 6132

Abstract

Impairment of mucociliary clearance with reduced airway fluid secretion leads to chronically inflamed airways. Cystic fibrosis transmembrane conductance regulator (CFTR) is crucially involved in airway fluid secretion and dexamethasone (dexa) has previously been shown to elevate CFTR activity in airway epithelial cells. However, [...] Read more.

Impairment of mucociliary clearance with reduced airway fluid secretion leads to chronically inflamed airways. Cystic fibrosis transmembrane conductance regulator (CFTR) is crucially involved in airway fluid secretion and dexamethasone (dexa) has previously been shown to elevate CFTR activity in airway epithelial cells. However, the pathway by which dexa increases CFTR activity is largely unknown. We aimed to determine whether the increase of CFTR activity by dexa is achieved by non-genomic signaling and hypothesized that the phosphoinositide 3-kinase (PI3K) pathway is involved in CFTR stimulation. Primary rat airway epithelial cells and human bronchial submucosal gland-derived Calu-3 cells were analyzed in Ussing chambers and kinase activation was determined by Western blots. Results demonstrated a critical involvement of PI3K and protein kinase B (AKT) signaling in the dexa-induced increase of CFTR activity, while serum and glucocorticoid dependent kinase 1 (SGK1) activity was not essential. We further demonstrated a reduced neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L) ubiquitin E3 ligase activity induced by dexa, possibly responsible for the elevated CFTR activity. Finally, increases of CFTR activity by dexa were demonstrated within 30 min accompanied by rapid activation of AKT. In conclusion, dexa induces a rapid stimulation of CFTR activity which depends on PI3K/AKT signaling in airway epithelial cells. Glucocorticoids might thus represent, in addition to their immunomodulatory actions, a therapeutic strategy to rapidly increase airway fluid secretion. Full article

(This article belongs to the Special Issue Physiological and Pathological Roles of ABC Transporters)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Phosphoinositide 3-kinase (PI3K) contributes to the increased cystic fibrosis transmembrane conductance regulator (CFTR) activity induced by dexa. Primary airway epithelial cells were treated with 100 nM dexa and LY-294002 (10 µM) for 24 h. Data bars represent mean + standard error of the mean (SEM) of ISC. Dexa increased the CFTRinh172-sensitive ISC. Addition of LY-294002 prevented the dexa-induced increase of CFTR activity (n = 20–25, ** p < 0.01, *** p < 0.001, analysis of variance (ANOVA) with Tukey’s post hoc test). Full article ">Figure 2
Serum and glucocorticoid dependent kinase 1 activity is not involved in the dexa-stimulated CFTR activity. Cells were treated with 100 nM dexa and GSK650394 (10 µM) for 24 h. Data bars represent mean + SEM of ISC. (a) In primary airway epithelial cells, the CFTRinh172-sensitive ISC was increased by dexa. Addition of GSK650394 displayed no effect on the dexa-induced increase of CFTR activity (n = 10–15, * p < 0.05, ** p < 0.01, ANOVA with Tukey’s post hoc test); (b) in Calu-3 cells CFTRinh172-sensitive ISC was increased by dexa. In agreement, GSK650394 displayed no effect on the dexa-induced increase of CFTR activity (n = 24–31, * p < 0.05, ** p < 0.01, ANOVA with Tukey’s post hoc test). Full article ">Figure 3
SGK1 activity is elevated by dexa and reduced by GSK650394. (a) Normalized densitometric evaluation of n-myc downregulated gene 1 (NDRG1) and pNDRG1 Western blots. Calu-3 cells were treated with 100 nM dexa and GSK650394 (10 µM) or mifepristone (10 µM) for 24 h (n = 4; * p < 0.05 by t-test); (b) western blot of pNDRG1 and total NDRG1 resulted in bands of 46 and 48 kDa. Full article ">Figure 4
Akt1/2 kinase inhibitor prevents the increase of CFTR activity induced by dexa. Cells were treated with 100 nM dexa and Akt1/2 kinase inhibitor (10 µM) for 24 h. Data bars represent the mean + SEM of ISC. (a) In primary airway epithelial cells, the CFTRinh172-sensitive ISC was increased by dexa. Addition of Akt1/2 kinase inhibitor prevented the dexa-induced increase of CFTR activity (n = 11–14; *** p < 0.001, ANOVA with Tukey’s post hoc test); (b) in Calu-3 cells the CFTRinh172-sensitive ISC was increased by dexa. Likewise, addition of Akt1/2 kinase inhibitor prevented the dexa-induced increase of CFTR activity (n = 36–37, *** p < 0.001, ANOVA with Tukey’s post hoc test). Full article ">Figure 5
AKT activity is elevated by dexa. (a) Normalized densitometric evaluation of pAKT and AKT Western blots. Calu-3 cells were treated with 100 nM dexa and Akt1/2 kinase inhibitor (10 µM) or mifepristone (10 µM) for 24 h. Addition of mifepristone and Akt1/2 kinase inhibitor blocked the increased AKT activity induced by dexa (n = 4; * p < 0.05; *** p < 0.001 by t-test); (b) western blot of pAKT and total AKT resulted in bands of 60 kDa. Full article ">Figure 6
Dexa increases phosphorylation of neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L). (a) Normalized densitometric evaluation of pNEDD4L, NEDD4L and α-tubulin Western blots. Calu-3 cells were treated with 100 nM dexa and mifepristone (10 µM) for 24 h (n = 5; * p < 0.05 by t-test); (b) western blot of pNEDD4L and total NEDD4L resulted in bands of 110 and 135 kDa. Western blot of α-tubulin resulted in bands of 52 kDa; (c) normalized densitometric evaluation of pNEDD4L, NEDD4L and α-tubulin Western blots. Calu-3 cells were treated with 100 nM dexa and Akt1/2 kinase inhibitor (10 µM) or GSK650394 (10 µM) for 24 h (n = 5; * p < 0.05 by t-test); (d) western blot of pNEDD4L and total NEDD4L resulted in bands of 110 and 135 kDa. Western blot of α-tubulin resulted in bands of 52 kDa. Full article ">Figure 7
Rapid effects of dexa on CFTR activity. Calu-3 cells were treated with 100 nM dexa for 30 min. Data bars represent the mean + SEM. (a) The CFTRinh172-sensitive ISC was significantly increased by dexa (n = 60, * p < 0.05 by t-test with Welch’s correction); (b) CFTR mRNA expression was not affected by dexa (n = 6); (c) normalized densitometric evaluation of pAKT and AKT Western blots (n = 3; * p < 0.05 by t-test). Western blot of pAKT and total AKT resulted in bands of 60 kDa; (d) normalized densitometric evaluation of pNDRG1 and NDRG1 Western blots (n = 3; * p < 0.05 by t-test). Western blot of pNDRG1 and total NDRG1 resulted in bands of 46 and 48 kDa; (e) normalized densitometric evaluation of pNEDD4L, NEDD4L and α-tubulin Western blots (n = 3; * p < 0.05 by t-test). Western blot of pNEDD4L and total NEDD4L resulted in bands of 110 and 135 kDa. Western blot of α-tubulin resulted in bands of 52 kDa. Full article ">

2039 KiB

Open AccessArticle

A Comparison of Lysosomal Enzymes Expression Levels in Peripheral Blood of Mild- and Severe-Alzheimer’s Disease and MCI Patients: Implications for Regenerative Medicine Approaches

by Francesco Morena, Chiara Argentati, Rosa Trotta, Lucia Crispoltoni, Anna Stabile, Alessandra Pistilli, Angela Di Baldassarre, Riccardo Calafiore, Pia Montanucci, Giuseppe Basta, Anna Pedrinolla, Nicola Smania, Massimo Venturelli, Federico Schena, Fabio Naro, Carla Emiliani, Mario Rende and Sabata Martino

Int. J. Mol. Sci. 2017, 18(8), 1806; https://doi.org/10.3390/ijms18081806 - 19 Aug 2017

Cited by 38 | Viewed by 5934

Abstract

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer’s Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive [...] Read more.

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer’s Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (β-Hexosaminidase, β-Galctosidase, β-Galactosylcerebrosidase, β-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of β-Galctosidase (Gal), β-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of Aβ-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD. Full article

(This article belongs to the Special Issue Molecular Research on Neurodegenerative Diseases)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
PBMCs from AD patients have primary hallmark of the disease: (A) Representative Western blotting of β-amyloid and Tau proteins analysis in PBMCs of mild AD, severe AD, MCI, and control subjects; (B,C) Densitometry analyses of both proteins were performed by the Fiji software (see Methods for details) and are reported as ratio toward β-actin protein, selected as reference. Each histogram is the mean ± SEM (standard error of the mean) of all samples analyzed in each group: CTR (n = 13); MCI (n = 10); mild AD (n = 19); and severe AD (n = 17); (D,E) Levels of β-amyloid and Tau proteins were determined in all samples of control group, MCI, mild AD and severe AD patients by using the ELISA assay. Results were expressed as the mean ± SEM of three independent experiments, each in triplicates. *** p < 0.001. Full article ">Figure 2
Lysosomal hydrolases activity (A–E) in PBMCs of mild AD, severe AD, MCI, and control subjects. Levels of hydrolases were measured by using specific fluorogenic substrates (see <a href="#sec4-ijms-18-01806" class="html-sec">Section 4</a> for details). Results were expressed as the mean ± SEM of five independent experiments, each in triplicates. * p < 0.05, ** p < 0.01, **** p < 0.0001. Full article ">Figure 3
Lysosomal proteases expression (A–D) in PBMCs of mild AD, severe AD, MCI, and control subjects. Levels of proteases were determined by Western blotting. Shown are the densitometry measurements of all analyses. Results were expressed as the mean ± SEM of five independent experiments, each in triplicates. * p < 0.05. Full article ">Figure 4
Lysosomal Hydrolases activity (A–E) in plasma of mild AD, severe AD, MCI, and control subjects. Levels of hydrolases were measured by using specific fluorogenic substrates (see <a href="#sec4-ijms-18-01806" class="html-sec">Section 4</a> for details). Results were expressed as the mean ± SEM of five independent experiments, each in triplicates. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 5
Lysosomal proteases expression (A–D) in plasma of mild AD, severe AD, MCI, and control subjects. Levels of proteases were determined in non-AD control group, mild AD and severe AD patients by using ELISA assay (see the Method section for details). Results were expressed as the mean ± SEM of five independent experiments, each in triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 6
Lysosomal enzymes are able to discriminate mild AD, severe AD, MCI, and control subjects. The Venn diagram illustrated the relationship between mild AD, severe AD, and MCI groups, where lysosomal enzymes showed statistical variation with respect to control group (A–D). Full article ">

4050 KiB

Open AccessArticle

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

by Tomohiko C. Umei, Hiroyuki Yamakawa, Naoto Muraoka, Taketaro Sadahiro, Mari Isomi, Sho Haginiwa, Hidenori Kojima, Shota Kurotsu, Fumiya Tamura, Rina Osakabe, Hidenori Tani, Kaori Nara, Hiroyuki Miyoshi, Keiichi Fukuda and Masaki Ieda

Int. J. Mol. Sci. 2017, 18(8), 1805; https://doi.org/10.3390/ijms18081805 - 19 Aug 2017

Cited by 19 | Viewed by 7563

Abstract

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms [...] Read more.

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming. Full article

(This article belongs to the Special Issue Stem Cell Research)

► Show Figures

Graphical abstract

615 KiB

Open AccessReview

FANCD2 and DNA Damage

by Manoj Nepal, Raymond Che, Chi Ma, Jun Zhang and Peiwen Fei

Int. J. Mol. Sci. 2017, 18(8), 1804; https://doi.org/10.3390/ijms18081804 - 19 Aug 2017

Cited by 46 | Viewed by 9597

Abstract

Investigators have dedicated considerable effort to understanding the molecular basis underlying Fanconi Anemia (FA), a rare human genetic disease featuring an extremely high incidence of cancer and many congenital defects. Among those studies, FA group D2 protein (FANCD2) has emerged as the focal [...] Read more.

Investigators have dedicated considerable effort to understanding the molecular basis underlying Fanconi Anemia (FA), a rare human genetic disease featuring an extremely high incidence of cancer and many congenital defects. Among those studies, FA group D2 protein (FANCD2) has emerged as the focal point of FA signaling and plays crucial roles in multiple aspects of cellular life, especially in the cellular responses to DNA damage. Here, we discuss the recent and relevant studies to provide an updated review on the roles of FANCD2 in the DNA damage response. Full article

(This article belongs to the Special Issue DNA Injury and Repair Systems)

► Show Figures

Figure 1

305 KiB

Open AccessReview

Platelet Aggregometry Testing: Molecular Mechanisms, Techniques and Clinical Implications

by Katalin Koltai, Gabor Kesmarky, Gergely Feher, Antal Tibold and Kalman Toth

Int. J. Mol. Sci. 2017, 18(8), 1803; https://doi.org/10.3390/ijms18081803 - 18 Aug 2017

Cited by 91 | Viewed by 11813

Abstract

Platelets play a fundamental role in normal hemostasis, while their inherited or acquired dysfunctions are involved in a variety of bleeding disorders or thrombotic events. Several laboratory methodologies or point-of-care testing methods are currently available for clinical and experimental settings. These methods describe [...] Read more.

Platelets play a fundamental role in normal hemostasis, while their inherited or acquired dysfunctions are involved in a variety of bleeding disorders or thrombotic events. Several laboratory methodologies or point-of-care testing methods are currently available for clinical and experimental settings. These methods describe different aspects of platelet function based on platelet aggregation, platelet adhesion, the viscoelastic properties during clot formation, the evaluation of thromboxane metabolism or certain flow cytometry techniques. Platelet aggregometry is applied in different clinical settings as monitoring response to antiplatelet therapies, the assessment of perioperative bleeding risk, the diagnosis of inherited bleeding disorders or in transfusion medicine. The rationale for platelet function-driven antiplatelet therapy was based on the result of several studies on patients undergoing percutaneous coronary intervention (PCI), where an association between high platelet reactivity despite P2Y12 inhibition and ischemic events as stent thrombosis or cardiovascular death was found. However, recent large scale randomized, controlled trials have consistently failed to demonstrate a benefit of personalised antiplatelet therapy based on platelet function testing. Full article

(This article belongs to the Special Issue Mechanisms of Platelet Thrombus Formation)

2284 KiB

Open AccessArticle

EpCAM Expression in Lymph Node Metastases of Urothelial Cell Carcinoma of the Bladder: A Pilot Study

by Christa A. M. Van der Fels, Stefano Rosati and Igle J. De Jong

Int. J. Mol. Sci. 2017, 18(8), 1802; https://doi.org/10.3390/ijms18081802 - 18 Aug 2017

Cited by 11 | Viewed by 5282

Abstract

In this retrospective pilot study, the feasibility of the epithelial cell adhesion molecule (EpCAM) as an imaging target for lymph node (LN) metastatic disease of urothelial cell carcinoma (UCC) of the bladder was investigated. LN metastases and LNs without metastases of patients who [...] Read more.

In this retrospective pilot study, the feasibility of the epithelial cell adhesion molecule (EpCAM) as an imaging target for lymph node (LN) metastatic disease of urothelial cell carcinoma (UCC) of the bladder was investigated. LN metastases and LNs without metastases of patients who underwent pelvic lymph node dissection because of muscle invasive bladder cancer (MIBC) were used. Primary tumors of the same patients were used from cystectomy specimen, transurethral resections, and biopsies. A pathologist, blinded to clinical data, scored EpCAM immunoreactivity. This method determines a total immunostaining score, which is the product of a proportion score and an intensity score. EpCAM expression was observed in 19/20 (95%) LNs with UCC metastases and in 11/12 (92%) of the primary tumors. EpCAM expression was absent in 14/14 (100%) LNs without metastases. Median EpCAM expression (TIS) in LN metastases was 5 (IQR 2.0–8.0) and in the primary tumors 6 (IQR 2.3–11.0). Based on the absence of staining in LNs without metastases, EpCAM show high tumor distinctiveness. EpCAM seems to be a feasible imaging target in LN metastases of UCC of the bladder. Pre- and perioperative visualization of these metastases will improve disease staging and improve the complete resection of LN metastases in MIBC. Full article

(This article belongs to the Special Issue Molecular Research on Urology)

► Show Figures

Figure 1

2620 KiB

Open AccessArticle

Enhanced Susceptibility of Ogg1 Mutant Mice to Multiorgan Carcinogenesis

by Anna Kakehashi, Naomi Ishii, Takahiro Okuno, Masaki Fujioka, Min Gi and Hideki Wanibuchi

Int. J. Mol. Sci. 2017, 18(8), 1801; https://doi.org/10.3390/ijms18081801 - 18 Aug 2017

Cited by 15 | Viewed by 4682

Abstract

The role of deficiency of oxoguanine glycosylase 1 (Ogg1) Mmh homolog, a repair enzyme of the 8-hydroxy-2’-deoxyguanosine (8-OHdG) residue in DNA, was investigated using the multiorgan carcinogenesis bioassay in mice. A total of 80 male and female six-week-old mice of C57BL/6J [...] Read more.

The role of deficiency of oxoguanine glycosylase 1 (Ogg1) Mmh homolog, a repair enzyme of the 8-hydroxy-2’-deoxyguanosine (8-OHdG) residue in DNA, was investigated using the multiorgan carcinogenesis bioassay in mice. A total of 80 male and female six-week-old mice of C57BL/6J background carrying a mutant Mmh allele of the Mmh/Ogg1 gene (Ogg1⁻^/⁻) and wild type (Ogg1^+/+) mice were administered N-diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), N-bis (2-hydroxypropyl) nitrosamine (DHPN) and 1,2-dimethylhydrazine dihydrochloride (DMH) (DMBDD) to induce carcinogenesis in multiple organs, and observed up to 34 weeks. Significant increase of lung adenocarcinomas incidence was observed in DMBDD-treated Ogg1⁻^/⁻ male mice, but not in DMBDD-administered Ogg1^+/+ animals. Furthermore, incidences of lung adenomas were significantly elevated in both Ogg1⁻^/⁻ males and females as compared with respective Ogg1⁻^/⁻ control and DMBDD-treated Ogg1^+/+ groups. Incidence of total liver tumors (hepatocellular adenomas, hemangiomas and hemangiosarcomas) was significantly higher in the DMBDD-administered Ogg1⁻^/⁻ males and females. In addition, in DMBDD-treated male Ogg1⁻^/⁻ mice, incidences of colon adenomas and total colon tumors showed a trend and a significant increase, respectively, along with significant rise in incidence of simple hyperplasia of the urinary bladder, and a trend to increase for renal tubules hyperplasia in the kidney. Furthermore, incidence of squamous cell hyperplasia in the forestomach of DMBDD-treated Ogg1⁻^/⁻ male mice was significantly higher than that of Ogg1^+/+ males. Incidence of small intestine adenomas in DMBDD Ogg1⁻^/⁻ groups showed a trend for increase, as compared to the wild type mice. The current results demonstrated increased susceptibility of Ogg1 mutant mice to the multiorgan carcinogenesis induced by DMBDD. The present bioassay could become a useful tool to examine the influence of various targets on mouse carcinogenesis. Full article

(This article belongs to the Special Issue Chemically-Induced DNA Damage, Mutagenesis, and Cancer)

► Show Figures

Graphical abstract

1705 KiB

Open AccessArticle

Determination of Sphingosine-1-Phosphate in Human Plasma Using Liquid Chromatography Coupled with Q-Tof Mass Spectrometry

by Emmanuel E. Egom, Ross Fitzgerald, Rebecca Canning, Rebabonye B. Pharithi, Colin Murphy and Vincent Maher

Int. J. Mol. Sci. 2017, 18(8), 1800; https://doi.org/10.3390/ijms18081800 - 18 Aug 2017

Cited by 10 | Viewed by 6139

Abstract

Evidence suggests that high-density lipoprotein (HDL) components distinct from cholesterol, such as sphingosine-1-phosphate (S1P), may account for the anti-atherothrombotic effects attributed to this lipoprotein. The current method for the determination of plasma levels of S1P as well as levels associated with HDL particles [...] Read more.

Evidence suggests that high-density lipoprotein (HDL) components distinct from cholesterol, such as sphingosine-1-phosphate (S1P), may account for the anti-atherothrombotic effects attributed to this lipoprotein. The current method for the determination of plasma levels of S1P as well as levels associated with HDL particles is still cumbersome an assay method to be worldwide practical. Recently, a simplified protocol based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific quantification of plasma levels of S1P with good accuracy has been reported. This work utilized a triple quadrupole (QqQ)-based LC-MS/MS system. Here we adapt that method for the determination of plasma levels of S1P using a quadrupole time of flight (Q-Tof) based LC-MS system. Calibration curves were linear in the range of 0.05 to 2 µM. The lower limit of quantification (LOQ) was 0.05 µM. The concentration of S1P in human plasma was determined to be 1 ± 0.09 µM (n = 6). The average accuracy over the stated range of the method was found to be 100 ± 5.9% with precision at the LOQ better than 10% when predicting the calibration standards. The concentration of plasma S1P in the prepared samples was stable for 24 h at room temperature. We have demonstrated the quantification of plasma S1P using Q-Tof based LC-MS with very good sensitivity, accuracy, and precision that can used for future studies in this field. Full article

(This article belongs to the Section Biochemistry)

► Show Figures

Figure 1

Figure 1
Representative extracted ion chromatogram (EIC) at m/z 380.2560 corresponding to the [M + H]+ ion of sphingosine-1-phosphate (S1P). S1P elution time and peak shape are illustrated. The retention time corresponds to that determined for the standards. Full article ">Figure 2
Comparison between the Waters X-Select CSH column, as used previously [<a href="#B32-ijms-18-01800" class="html-bibr">32</a>] (Red), and a Phenomenex Kinetex EVO C18 used here (Green). Full article ">Figure 3
(A) When only the ions of interest are extracted [M+H]+, both mass accuracy and isotope pattern are consistent with the presence of S1P. The black lines are the spectrum overlaid with predicted isotope pattern; (B) Representative raw spectrum extracted under the S1P peak with background subtracted. The evidence of blood proteins (probably serum albumin) co-eluting is shown by the peaks in the charge envelope between ~700 and 1500 m/z. Full article ">Figure 4
1/x weighted calibration curve recommended to be used. Black dots: calibration standards; blue triangles: QC samples; pink arrow: denotes human plasma. Black dots represent calibration standards; blue triangles are QC samples and pink arrow is a human plasma sample. Full article ">Figure 5
Non-weighted linear regression calibration curve of three injection replicates at seven calibration levels (4 to 10) (0.05 to 4.5 µM) with very good correlation and linearity over the entire range. The heteroscedasticity of the data is clear with absolute variance increasing with concentration. Black dots represent calibration standards; blue triangles are QC samples. Full article ">Figure 6
The data is heteroscedastic and would be better modelled using a weighted linear regression. Plot of the positive and negative standard deviation against calibration levels show increased absolute variation with increasing concentration. Full article ">Figure 7
Response/concentration ratio is constant from 1.5 to 4.5 µM with a decline noticeable at 0.05 µM. Full article ">Figure 8
The consistency of the signal over greater than 12 h and 50 injections demonstrates the stability of the instrument and the samples. (Quality control and calibration injections were performed between the above replicates). Full article ">

3575 KiB

Open AccessReview

Aquaporin-4 Functionality and Virchow-Robin Space Water Dynamics: Physiological Model for Neurovascular Coupling and Glymphatic Flow

by Tsutomu Nakada, Ingrid L. Kwee, Hironaka Igarashi and Yuji Suzuki

Int. J. Mol. Sci. 2017, 18(8), 1798; https://doi.org/10.3390/ijms18081798 - 18 Aug 2017

Cited by 61 | Viewed by 13731

Abstract

The unique properties of brain capillary endothelium, critical in maintaining the blood-brain barrier (BBB) and restricting water permeability across the BBB, have important consequences on fluid hydrodynamics inside the BBB hereto inadequately recognized. Recent studies indicate that the mechanisms underlying brain water dynamics [...] Read more.

The unique properties of brain capillary endothelium, critical in maintaining the blood-brain barrier (BBB) and restricting water permeability across the BBB, have important consequences on fluid hydrodynamics inside the BBB hereto inadequately recognized. Recent studies indicate that the mechanisms underlying brain water dynamics are distinct from systemic tissue water dynamics. Hydrostatic pressure created by the systolic force of the heart, essential for interstitial circulation and lymphatic flow in systemic circulation, is effectively impeded from propagating into the interstitial fluid inside the BBB by the tightly sealed endothelium of brain capillaries. Instead, fluid dynamics inside the BBB is realized by aquaporin-4 (AQP-4), the water channel that connects astrocyte cytoplasm and extracellular (interstitial) fluid. Brain interstitial fluid dynamics, and therefore AQP-4, are now recognized as essential for two unique functions, namely, neurovascular coupling and glymphatic flow, the brain equivalent of systemic lymphatics. Full article

(This article belongs to the Special Issue Cerebral Blood Flow and Metabolism)

► Show Figures

Graphical abstract

3786 KiB

Open AccessArticle

Immune-Response Patterns and Next Generation Sequencing Diagnostics for the Detection of Mycoses in Patients with Septic Shock—Results of a Combined Clinical and Experimental Investigation

by Sebastian O. Decker, Annette Sigl, Christian Grumaz, Philip Stevens, Yevhen Vainshtein, Stefan Zimmermann, Markus A. Weigand, Stefan Hofer, Kai Sohn and Thorsten Brenner

Int. J. Mol. Sci. 2017, 18(8), 1796; https://doi.org/10.3390/ijms18081796 - 18 Aug 2017

Cited by 47 | Viewed by 13033

Abstract

Fungi are of increasing importance in sepsis. However, culture-based diagnostic procedures are associated with relevant weaknesses. Therefore, culture- and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of β-d-glucan, interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin [...] Read more.

Fungi are of increasing importance in sepsis. However, culture-based diagnostic procedures are associated with relevant weaknesses. Therefore, culture- and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of β-d-glucan, interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, -4, -6, -10, -17A, and mid-regional proadrenomedullin (MR-proADM) were evaluated in 50 septic patients at six consecutive time points within 28 days after sepsis onset. Furthermore, immune-response patterns during infections with Candida spp. were studied in a reconstituted human epithelium model. In total, 22% (n = 11) of patients suffered from a fungal infection. An NGS-based diagnostic approach appeared to be suitable for the identification of fungal pathogens in patients suffering from fungemia as well as in patients with negative blood cultures. Moreover, MR-proADM and IL-17A in plasma proved suitable for the identification of patients with a fungal infection. Using RNA-seq., adrenomedullin (ADM) was shown to be a target gene which is upregulated early after an epithelial infection with Candida spp. In summary, an NGS-based diagnostic approach was able to close the diagnostic gap of routinely used culture-based diagnostic procedures, which can be further facilitated by plasmatic measurements of MR-proADM and IL-17A. In addition, ADM was identified as an early target gene in response to epithelial infections with Candida spp. Full article

(This article belongs to the Special Issue Sepsis)

► Show Figures

Graphical abstract

1662 KiB

Open AccessArticle

Rho-Kinase Blockade Attenuates Podocyte Apoptosis by Inhibiting the Notch Signaling Pathway in Diabetic Nephropathy

by Keiichiro Matoba, Daiji Kawanami, Yosuke Nagai, Yusuke Takeda, Tomoyo Akamine, Sho Ishizawa, Yasushi Kanazawa, Tamotsu Yokota and Kazunori Utsunomiya

Int. J. Mol. Sci. 2017, 18(8), 1795; https://doi.org/10.3390/ijms18081795 - 18 Aug 2017

Cited by 36 | Viewed by 6078

Abstract

Podocyte apoptosis is a key process in the onset of diabetic nephropathy. A significant body of evidence shows that the Notch signaling pathway plays a central role in this process. We found that Rho-kinase mediates transforming growth factor β (TGF-β)-induced Notch ligand Jag1 [...] Read more.

Podocyte apoptosis is a key process in the onset of diabetic nephropathy. A significant body of evidence shows that the Notch signaling pathway plays a central role in this process. We found that Rho-kinase mediates transforming growth factor β (TGF-β)-induced Notch ligand Jag1 expression. Importantly, TGF-β-mediated podocyte apoptosis was attenuated by Rho-kinase inhibition. Mechanistically, Rho-kinase regulated Jag1 induction via the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) but not Smad pathways. Consistently, the Rho-kinase inhibitor fasudil prevented albuminuria and the urinary excretion of nephrin in db/db mice and reduced the prevalence of podocyte apoptosis and Jag1 expression. Finally, the expression of Jag1 and apoptosis markers such as Bax and cyclin-dependent kinase inhibitor 1A (CDKN1A) was decreased in podocytes derived from db/db mice treated with fasudil. The present study provides evidence that Rho-kinase plays a key role in podocyte apoptosis. Rho-kinase is an attractive therapeutic target for diabetic nephropathy. Full article

(This article belongs to the Special Issue Advances in Chronic Kidney Disease 2017)

► Show Figures

Graphical abstract

Journal Menu

Journal Browser

Int. J. Mol. Sci., Volume 18, Issue 8 (August 2017) – 223 articles

Further Information

Guidelines

MDPI Initiatives

Follow MDPI