Abnormal extracellular matrix remodelling in the cervix of pregnant relaxin-deficient mice is not associated with reduced matrix metalloproteinase expression or activity

2018 ◽  
Vol 30 (9) ◽  
pp. 1214 ◽  
Author(s):  
Sarah A. Marshall ◽  
Jonathan T. McGuane ◽  
Yu May Soh ◽  
Helen M. Gehring ◽  
Emma Simpson ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Quantitative Polymerase Chain Reaction ◽  
Late Pregnancy ◽  
Mmp9 Expression ◽  
Extracellular Matrix Remodelling ◽  
Polymerase Chain ◽  
Collagen Remodelling ◽  
Esr1 Expression ◽  
Histological Staining ◽  
Relaxin Receptor

Relaxin regulates cervical extracellular matrix (ECM) remodelling during pregnancy by modifying collagen and other ECM molecules by unknown mechanisms. We hypothesised that abnormal collagen remodelling in the cervix of pregnant relaxin-deficient (Rln1−/−) mice is due to excessive collagen (Col1a1 and Col3a1) and decreased matrix metalloproteinases (Mmp2, Mmp9, Mmp13 and Mmp7) and oestrogen receptors (Esr1 and Esr2). Quantitative polymerase chain reaction, gelatinase zymography, MMP activity assays and histological staining evaluated changes in ECM in pregnant wildtype (Rln1+/+) and Rln1−/− mice. Cervical Col1a1, Col3a1 and total collagen increased in Rln1−/− mice and were higher at term compared with Rln1+/+ mice. This was not correlated with a decrease in gelatinase (Mmp2, Mmp9) expression or activity, Mmp7 or Mmp13 expression, which were all significantly higher in Rln1−/− mice. In late pregnancy, circulating MMP2 and MMP9 were unchanged. Esr1 expression was highest in Rln1+/+ and Rln1−/− mice in late pregnancy, coinciding with a decrease in Esr2 in Rln1+/+ but not Rln1−/− mice. The relaxin receptor (Rxfp1) decreased slightly in late-pregnant Rln1+/+ mice, but was significantly higher in Rln1−/− mice. In summary, relaxin deficiency results in increased cervical collagen in late pregnancy, which is not explained by a reduction in Mmp expression or activity or decreased Rxfp1. However, an imbalance between Esr1 and Esr2 may be involved.

scholarly journals Brain gene expression in a novel mouse model of postpartum mood disorder

2019 ◽  
Vol 10 (1) ◽  
pp. 168-174
Author(s):  
Trevor Humby ◽  
William Davies
Keyword(s):  
Gene Expression ◽  
Pathway Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Late Pregnancy ◽  
Maternal Behaviour ◽  
Aberrant Expression ◽  
Brain Gene Expression ◽  
False Discovery ◽  
Expression Activity ◽  
Polymerase Chain

Abstract Background Steroid sulfatase (STS) cleaves sulfate groups from steroid hormones; its expression/activity increases in late pregnancy and into the postpartum period. STS-deficient human and mouse mothers display elevated psychopathology and abnormal behaviour respectively; in mice, these effects can be partially normalised by antipsychotic (ziprasidone) administration. Methodology We compared brain gene expression in new mouse mothers administered the STS inhibitor 667-Coumate, or vehicle; significant changes were followed-up with pathway analysis and quantitative polymerase chain reaction (qPCR). Finally, the effects of combined 667-Coumate and ziprasidone administration on expression of the most robustly differentially-expressed genes were examined. Results Surprisingly, no between-group gene expression changes were detected at a False Discovery Rate (FDR)-corrected p<0.1. 1,081 unique expression changes were detected at p<0.05, two top hits were verified by qPCR, and pathway analysis indicated enrichment of genes involved in olfactory transduction. The expression of Stoml3 and Cyp2g1 was unaffected by ziprasidone administration. Conclusions Postpartum behavioural abnormalities in STS-deficient mothers are likely to be the culmination of many small gene expression changes. Our data are consistent with the idea that olfactory function is key to maternal behaviour in mice, and suggest that aberrant expression of olfactory system genes may underlie abnormal maternal behaviour in STS-deficient women.

Fibronectin Gene Up-regulation by Arnica montana in Human Macrophages: Validation by Real-Time Polymerase Chain Reaction Assay

Homeopathy ◽  
2020 ◽  
Vol 109 (03) ◽  
pp. 140-145
Author(s):  
Marta Marzotto ◽  
Fabio Arruda-Silva ◽  
Paolo Bellavite
Keyword(s):  
Extracellular Matrix ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Heparan Sulphate ◽  
Chain Reaction ◽  
Human Macrophages ◽  
Arnica Montana ◽  
Positive Effects ◽  
Polymerase Chain

Abstract Background and Aim Arnica montana L. (Arnica m.) is a popular traditional medicine, used for its therapeutic properties in healing traumas, but little is known about its biological action on tissue formation and repair. This new work tested the effects of Arnica m. homeopathic dilutions on human macrophages, key cells in tissue defence and repair. Materials and Methods Macrophages derived from the THP-1 cell line were differentiated with interleukin-4 to induce a ‘wound-healing’-like phenotype, and treated with various dilutions of Arnica m. centesimal (100 times) dilutions (2c, 3c, 5c, 9c, and 15c) or control solvent for 24 hours. RNA samples from cultured cells were analysed by real-time quantitative polymerase chain reaction in five separate experiments. Results Arnica montana at the 2c dilution (final concentration of sesquiterpene lactones in cell culture = 10−8 mol/L) significantly stimulated the expression of three genes which code for regulatory proteins of the extracellular matrix, namely FN1 (fibronectin 1, % increase of 21.8 ± standard error of the mean 4.6), low-density lipoprotein-receptor-related protein 1 (% increase of 33.4 ± 6.1) and heparan sulphate proteoglycan 2 (% increase of 21.6 ± 9.1). Among these genes, the most quantitatively expressed was FN1. In addition, FN1, unlike other candidate genes, was upregulated in cells treated with higher dilutions/dynamisations (3c, 5c, and 15c) of Arnica m. Conclusion The results support evidence that the extracellular matrix is a potential therapeutic target of Arnica m., with positive effects on cell adhesion and migration during tissue development and healing.

scholarly journals EZH2 Promotes Extracellular Matrix Degradation via Nuclear Factor-κB(NF-κB) and p38 signaling Pathways in pulpitis

2021 ◽  
Author(s):  
jie He ◽  
Man Qin ◽  
Yingyi Chen ◽  
Ziqi Hu ◽  
Ling Ye ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Polymerase Chain Reaction ◽  
Signaling Pathways ◽  
Dental Pulp ◽  
Quantitative Polymerase Chain Reaction ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Matrix Degradation ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background: Pulpitis is a complicated chronic inflammatory process which in a dynamic balance between damage and repair. Extracellular matrix plays an important regulatory role in wound healing and tissue repair. The aim of this study was to explore role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2) on the degradation of extracellular matrix during pulpitis. Methods : Quantitative polymerase chain reaction was used to assess the expression of matrix metalloproteinases (MMPs) and type Ⅰ collagen in HDPCs upon EZH2 and EI1 stimulation. The mechanism of EZH2 affecting extracellular matrix was explored through quantitative polymerase chain reaction and Western blot. A rat model of dental pulp inflammation was established, and the expression of type Ⅰ collagen in dental pulp under EZH2 stimulation was detected by immunohistochemical staining. Results :EZH2 upregulated the expression of MMP-1, MMP-3, MMP-8 and MMP-10 and decreased the production of type Ⅰ collagen in HDPCs, while EI1 had the opposite effect .EZH2 activated the Nuclear Factor-κB(NF-κB)and p38 signaling Pathways in HDPCs, the inhibition of which reversed the induction of MMPs and the suppression of type Ⅰ collagen .EZH2 can downregulated the type Ⅰ collagen levels in an experimental model of dental pulpitis in rats. Conclusion: EZH2 promotes extracellular matrix degradation via Nuclear Factor-κB(NF-κB)and P38 signaling pathways in pulpitis.EZH2 can decrease the type Ⅰ collagen levels in vivo and vitro.

scholarly journals EZH2 Promotes Extracellular Matrix Degradation via Nuclear Factor-kB(NF-kB)and p38 signaling Pathways in Pulpitis.

2020 ◽  
Author(s):  
jie He ◽  
Man Qin ◽  
Yingyi Chen ◽  
Ziqi Hu ◽  
Ling Ye ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Signaling Pathways ◽  
Dental Pulp ◽  
Type I Collagen ◽  
Quantitative Polymerase Chain Reaction ◽  
Type I ◽  
Nuclear Factor ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Nuclear Factor Kb

Abstract Background: Pulpitis is a complicated chronic inflammatory process which in a dynamic balance between damage and repair. Extracellular matrix plays an important regulatory role in wound healing and tissue repair. The aim of this study was to explore role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2) on the degradation of extracellular matrix during pulpitis. Methods : Quantitative polymerase chain reaction was used to assess the expression of matrix metalloproteinases (MMPs) and type I collagen in HDPCs upon EZH2 and EI1 stimulation. The mechanism of EZH2 affecting extracellular matrix was explored through quantitative polymerase chain reaction and Western blot. A rat model of dental pulp inflammation was established, and the expression of type I collagen in dental pulp under EZH2 stimulation was detected by immunohistochemical staining. Results :EZH2 upregulated the expression of MMP-1, MMP-3, MMP-8 and MMP-10 and decreased the production of type I collagen in HDPCs, while EI1 had the opposite effect .EZH2 activated the Nuclear Factor-kB(NF-kB)and p38 signaling Pathways in HDPCs, the inhibition of which reversed the induction of MMPs and the suppression of type I collagen .EZH2 can downregulated the type I collagen levels in an experimental model of dental pulpitis in rats. Conclusion: EZH2 promotes extracellular matrix degradation via Nuclear Factor-kB(NF-kB)and P38 signaling pathways in pulpitis.EZH2 can decrease the type I collagen levels in vivo and vitro.

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.

Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1759-1767 ◽  
Author(s):  
Kyu-Tae Kim ◽  
Kristin Baird ◽  
Joon-Young Ahn ◽  
Paul Meltzer ◽  
Michael Lilly ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Quantitative Polymerase Chain Reaction ◽  
Colony Growth ◽  
Dominant Negative ◽  
Internal Tandem Duplication ◽  
Downstream Target ◽  
Before And After ◽  
Flt3 Expression ◽  
Polymerase Chain ◽  
Expression Microarrays

AbstractConstitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling. (Blood. 2005;105: 1759-1767)

scholarly journals Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 352
Author(s):  
Wei Wei ◽  
Valeria Trivellone ◽  
Christopher H. Dietrich ◽  
Yan Zhao ◽  
Kristi D. Bottner-Parker ◽  
...  
Keyword(s):  
Host Range ◽  
Quantitative Polymerase Chain Reaction ◽  
Rflp Analysis ◽  
Natural Habitats ◽  
Genetic Lineages ◽  
Genetic Subgroup ◽  
Polymerase Chain ◽  
Phloem Feeding ◽  
Fresh Insight

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

scholarly journals Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

2021 ◽  
Author(s):  
Sara Keränen ◽  
Santeri Suutarinen ◽  
Rahul Mallick ◽  
Johanna P. Laakkonen ◽  
Diana Guo ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Rank Correlation ◽  
Inflammatory Cells ◽  
Vessel Remodeling ◽  
Brain Avm ◽  
Inflammatory Drugs ◽  
Polymerase Chain ◽  
The Brain ◽  
Cox2 Expression

Abstract Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

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