scholarly journals Active liver X receptor signaling in phagocytes in multiple sclerosis lesions

2017 ◽  
Vol 24 (3) ◽  
pp. 279-289 ◽  
Author(s):  
Jo Mailleux ◽  
Tim Vanmierlo ◽  
Jeroen FJ Bogie ◽  
Elien Wouters ◽  
Dieter Lütjohann ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Monocyte ◽  
Liver X Receptor ◽  
Spectrometric Analysis ◽  
Protein Levels ◽  
Myelin Phagocytosis ◽  
Polymerase Chain ◽  
Cholesterol Precursors ◽  
Chromatographic Mass

Objective: We sought to determine the liver X receptor (LXR) ligands present in human macrophages after myelin phagocytosis and whether LXRs are activated in multiple sclerosis (MS) lesions. Methods: We used real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry to determine expression of LXRs and their response genes in human phagocytes after myelin phagocytosis and in active MS lesions. We used gas chromatographic/mass spectrometric analysis to determine LXR-activating oxysterols and cholesterol precursors present and formed in myelin and myelin-incubated cells, respectively. Results: Myelin induced LXR response genes ABCA1 and ABCG1 in human monocyte-derived macrophages. In active MS lesions, we found that both gene expression and protein levels of ABCA1 and apolipoprotein E ( APOE) are upregulated in foamy phagocytes. Moreover, we found that the LXR ligand 27-hydroxycholesterol (27OHC) is significantly increased in human monocyte-derived macrophages after myelin uptake. Conclusion: LXR response genes are upregulated in phagocytes present in active MS lesions, indicating that LXRs are activated in actively demyelinating phagocytes. In addition, we have shown that myelin contains LXR ligands and that 27OHC is generated in human monocyte-derived macrophages after myelin processing. This suggests that LXRs in phagocytes in active MS lesions are activated at least partially by (oxy)sterols present in myelin and the generation thereof during myelin processing.

scholarly journals Decreased Expression of GPER1 Gene and Protein in Goiter

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Raquel Weber ◽  
Ana Paula Santin Bertoni ◽  
Laura Walter Bessestil ◽  
Ilma Simoni Brum ◽  
Tania Weber Furlanetto
Keyword(s):  
Estrogen Receptor ◽  
Quantitative Polymerase Chain Reaction ◽  
Thyroid Tissue ◽  
Chain Reaction ◽  
Normal Thyroid ◽  
Protein Levels ◽  
Protein Expressions ◽  
Polymerase Chain ◽  
G Protein Coupled

Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERαand ERβ) has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1), has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. In normal thyroid (n=16) and goiter (n=19), GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n=15) but in only 72% of goiter samples (n=13). When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

scholarly journals Total Versus Free Placental Growth Factor Levels in the Pathogenesis of Preeclampsia

Hypertension ◽  
2020 ◽  
Vol 76 (3) ◽  
pp. 875-883
Author(s):  
Edouard Lecarpentier ◽  
Zsuzsanna K. Zsengellér ◽  
Saira Salahuddin ◽  
Ambart E. Covarrubias ◽  
Agnes Lo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Ex Vivo ◽  
Quantitative Polymerase Chain Reaction ◽  
Placental Growth Factor ◽  
Protein Levels ◽  
Key Characteristics ◽  
Human Blood Samples ◽  
Polymerase Chain ◽  
Low Levels ◽  
Independent Cohort

Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF (placental growth factor), are key characteristics of preeclampsia. However, it is unclear if the low levels of plasma PlGF noted during preeclampsia are due to decreased placental production of PlGF or due to binding of PlGF by increased circulating sFLT-1. Here, we describe a biochemical procedure to dissociate PlGF-sFLT-1 complex ex vivo and when used in conjunction with an immunoassay platform, demonstrate a method to measure total and free PlGF in human blood samples. Using this method, we noted that plasma free PlGF levels were significantly lower in preeclampsia (N=22) than in nonhypertensive controls (N=24; mean, 314 versus 686 pg/mL, P <0.05), but total PlGF levels were not different (mean, 822 versus 800 pg/mL, P =0.49). In contrast, total sFLT-1 levels were significantly higher in preeclampsia than in nonhypertensive controls (mean, 16 957 versus 3029 pg/mL, P <0.01) and sFLT-1 levels correlated with bound PlGF levels (bound PlGF=total PlGF−free PlGF) in these samples ( r 2 =0.68). We confirmed these findings in an independent cohort of subjects (N=49). Furthermore, we did not detect any difference in PlGF mRNA by quantitative polymerase chain reaction or in PlGF protein expression by immunohistochemistry in preeclamptic placentas when compared with nonhypertensive controls. In contrast, sFLT-1 mRNA and protein levels were upregulated in placentas from women with preeclampsia. Taken together with prior studies, our results provide evidence that decrease in circulating PlGF noted during preeclampsia is largely mediated by excess circulating sFLT-1.

Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 863-873 ◽  
Author(s):  
Bartosz Kempisty ◽  
Hanna Piotrowska ◽  
Dorota Bukowska ◽  
Magdalena Woźna ◽  
Sylwia Ciesiółka ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Quantitative Polymerase Chain Reaction ◽  
Microscopic Analysis ◽  
Maturation Stage ◽  
Protein Distribution ◽  
Protein Levels ◽  
Peripheral Area ◽  
Before And After ◽  
Polymerase Chain

SummaryThis study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus–oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

scholarly journals ARG1 could be expressed by human, but not represent for repair

2020 ◽  
Author(s):  
Chenyu Chu ◽  
Chen Hu ◽  
Shengan Rung ◽  
Yufei Wang ◽  
Yili Qu ◽  
...  
Keyword(s):  
Bone Tissue ◽  
Quantitative Polymerase Chain Reaction ◽  
Healing Process ◽  
Tissue Samples ◽  
Protein Levels ◽  
Arginase 1 ◽  
Tissue Healing ◽  
Human Bone Tissue ◽  
Polymerase Chain ◽  
Clinical Healing

AbstractArginase 1 (ARG1), typically expressed under stress or stimulation rather than in the healthy condition in humans. However, more and more studies claim ARG1 could be expressed by human and represents healing process. Including THP-1 monocyte shift to M2 macrophages rendered a higher expression of ARG1 than that with bone-graft has an ideal effect on anti-inflammation, resulting in tissue healing. With the higher expression of ARG1 seemingly induced by the constructed material and represented by the anti-inflammatory situation. For these reasons, this study focuses on whether traditional markers of macrophages could be express by human or not during clinical healing process. Through harvesting bone tissue samples during bone healing process and confirm the expression of markers at transcriptome and protein levels. The results showed the expression of ARG1 relatively low by bulk-RNA sequencing, quantitative polymerase chain reaction (qPCR), immunofluorescence (IF) and flow cytometry or could not be detected during process of healing of healthy donor. Taken together, results indicate that the ARG1 is not chosen for a regenerative marker in human bone tissue.

Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells

2009 ◽  
Vol 877 (22) ◽  
pp. 2081-2086 ◽  
Author(s):  
Jure Acimovic ◽  
Anita Lövgren-Sandblom ◽  
Katalin Monostory ◽  
Damjana Rozman ◽  
Marko Golicnik ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mass Spectrometric ◽  
Plant Sterols ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Cholesterol Precursors ◽  
Chromatographic Mass

Effects of calcium hydroxide and N-acetylcysteine on MMP-2, MMP-9, TIMP-1 and TIMP-2 in LPS-stimulated macrophage cell lines

2017 ◽  
Vol 43 (6) ◽  
pp. 571-577
Author(s):  
Eda Ezgi Aslantaş ◽  
Yasemin Aksoy ◽  
Yeliz Zülfiye Akkaya Ulum ◽  
Deniz Ceyhan ◽  
Banu Peynircioglu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Calcium Hydroxide ◽  
Human Monocyte ◽  
Matrix Metalloproteinase 2 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Macrophage Cell ◽  
Protein Levels ◽  
Bonferroni Test ◽  
Polymerase Chain

Abstract Aim This study was evaluated the effects of N-acetylcysteine (NAC) and calcium hydroxide (Ca(OH)2) on the expression levels of matrix metalloproteinase -2, -9 (MMP-2, -9) and tissue inhibitor metalloproteinase -1, -2 (TIMP-1, -2) in lipopolysaccharide (LPS)-stimulated human macrophages. Methods Human monocyte precursor cells (THP-1) were differentiated into macrophage-adherent cells and were stimulated with LPS for 24 h. Then individually incubated with NAC or Ca(OH)2 for 24, 48 and 72 h. Following incubation, protein expression and mRNA levels of MMP-2, -9 and TIMP-1, -2 were evaluated using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Data were statistically analysed using two-way ANOVA, to followed by Bonferroni test at α=0.05. Results NAC significantly decreased mRNA expression and protein levels of MMP-9, while Ca(OH)2 decreased mRNA expression alone at 24 h. NAC and Ca(OH)2 decreased mRNA expression of MMP-2 at 24 h, while NAC increased this expression at 48 h. Although NAC and Ca(OH)2 decreased the mRNA expression of TIMP-1, -2 at 24 h, only NAC increased mRNA expression of TIMP-1 at 48 h. Conclusion At the early stages of inflammation, NAC and Ca(OH)2 have anti-inflammatory effects on macrophages.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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