scholarly journals Type III Collagen is Required for Adipogenesis and Actin Stress Fibre Formation in 3T3-L1 Preadipocytes

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 156
Author(s):  
Mohammad Al Hasan ◽  
Patricia E. Martin ◽  
Xinhua Shu ◽  
Steven Patterson ◽  
Chris Bartholomew
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Type Iii Collagen ◽  
Type Iii ◽  
Actin Stress Fibre ◽  
Matrix Gene ◽  
Gene Transcripts ◽  
Polymerase Chain ◽  
Oil Red O Staining ◽  
Oil Red O

GPR56 is required for the adipogenesis of preadipocytes, and the role of one of its ligands, type III collagen (ColIII), was investigated here. ColIII expression was examined by reverse transcription quantitative polymerase chain reaction, immunoblotting and immunostaining, and its function investigated by knockdown and genome editing in 3T3-L1 cells. Adipogenesis was assessed by oil red O staining of neutral cell lipids and production of established marker and regulator proteins. siRNA-mediated knockdown significantly reduced Col3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. Col3a1−/− 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ2 (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). Col3a1−/− 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin (Fn) and collagen (Col4a1, Col6a1) extracellular matrix gene transcripts. Col3a1−/− 3T3-L1 cells also had dramatically reduced actin stress fibres. We conclude that ColIII is required for 3T3-L1 preadipocyte adipogenesis as well as the formation of actin stress fibres. The phenotype of Col3a1−/− 3T3-L1 cells is very similar to that of Gpr56−/− 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.

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.

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 C/EBPα directs monocytic commitment of primary myeloid progenitors

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1223-1229 ◽  
Author(s):  
Dehua Wang ◽  
Jenice D'Costa ◽  
Curt I. Civin ◽  
Alan D. Friedman
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Fold Increase ◽  
Pcr Assay ◽  
Gm Csf ◽  
Specific Effects ◽  
Proliferation And Apoptosis ◽  
Polymerase Chain ◽  
Lineage Markers ◽  
Transcriptional Induction

Abstract C/EBPα is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBPα in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBPα-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradiol reduced activated C/EBPα-ER to near the physiologic range. C/EBPα-ER increased Mac1+/Gr1–/MPO–/low monocytes 1.9-fold while reducing Mac1+/Gr1+/MPOhi granulocytes 2.5-fold at 48 hours, even in 0.01 μM estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transduced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in IL-3/IL-6/SCF or GM-CSF, but not G-CSF, even in 0.01 μM estradiol. C/EBPα-ER induced PU.1 mRNA, and PU.1-ER stimulated monocytic development, suggesting that transcriptional induction of PU.1 by C/EBPα contributes to monopoiesis. A C/EBPα variant incapable of zippering with c-Jun did not induce monopoiesis, and a variant unable to bind NF-κB p50 stimulated granulopoiesis, suggesting their cooperation with C/EBPα during monocytic commitment.

Sinonasal Surfactant Protein A1, A2, and D Gene Expression in Cystic Fibrosis: A Preliminary Report

2007 ◽  
Vol 137 (1) ◽  
pp. 34-38 ◽  
Author(s):  
Bradford A. Woodworth ◽  
Rachel Wood ◽  
John E. Baatz ◽  
Rodney J. Schlosser
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Nasal Polyposis ◽  
Quantitative Polymerase Chain Reaction ◽  
Surfactant Protein ◽  
Fungal Rhinosinusitis ◽  
Allergic Fungal Rhinosinusitis ◽  
Novel Treatment ◽  
Polymerase Chain

OBJECTIVE: To measure alterations in SPA1, A2, and D gene expression in various forms of inflammatory chronic rhinosinusitis (CRS). STUDY DESIGN AND SETTING: Sinus mucosal biopsies were performed in patients with allergic fungal rhinosinusitis (AFS), CRS with nasal polyposis, cystic fibrosis (CF), and controls. SP mRNA was measured with quantitative polymerase chain reaction. RESULTS: Patients with CF (n = 4) showed significantly increased SPA1 (82-fold), SPA2 (100-fold), and SPD (47-fold) mRNA ( P < 0.05) when compared with controls (n = 5). Patients with CRS with nasal polyposis (n = 5) also demonstrated elevated SPA1 (27-fold), SPA2 (13-fold), and SPD (13-fold). Patients with AFS (n = 7) had increased SPA1 (5-fold), SPA2 (9-fold), and SPD (17-fold), but were not statistically significant. CONCLUSION: SPA1, A2, and D are upregulated in various forms of CRS, but are significantly elevated in cystic fibrosis CRS. SIGNIFICANCE: Understanding the role of SPs in CRS will help develop novel treatment approaches for sinonasal pathoses.

Amino acid Sequence of a Peptide from Type III Collagen Involved in Platelet Adhesion

1979 ◽  
Author(s):  
F. Fauvel ◽  
Y.J. Legrand ◽  
H. Bentz ◽  
G. Pignaud ◽  
K. Kühn ◽  
...  
Keyword(s):  
Vessel Wall ◽  
Cyanogen Bromide ◽  
Platelet Adhesion ◽  
Type Iii Collagen ◽  
Salt Precipitation ◽  
Type Iii ◽  
Imino Acid ◽  
Precipitation Type ◽  
Adhesion Of Platelets

The probable importance of the role of type III collagen in the initiation of thrombosis is due to its localization in the subendothelial layers of the vessel wall.The adhesion of platelets to type III has been therefore quantified by a method based on the filtration of non-adhesive 14C 5HT-labe11ed platelets through a Sepharose 2B column.Type III collagen was purified from calf skin by pepsin extraction and salt precipitation. Type III collagen was cleaved by cyanogen bromide and the adhesion induced by the resulting peptides was measured. The activity was attached to the central alpha l(III) CB4 peptide which was further cleaved by hydroxy lamine, chymotrypsin and trypsin. An adhesive potency was linked to three fragments (HA 1 obtained by hydroxylamine, C2 by chymotrypsin and T2 by trypsin) which possess a common portion of 9 amino-acids, localized in the central part of the alpha l(III) CB4 peptide and of the entire alpha 1(III) Chains, which probably represents the active part of type III collagen. Its activity could be due to a particular sterical conformation linked to the presence of 3 imino acid residues in the sequence Gly-Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys of this fragment.

scholarly journals Thrombotic, inflammatory, and HIF-regulated genes and thrombosis risk in polycythemia vera and essential thrombocythemia

2020 ◽  
Vol 4 (6) ◽  
pp. 1115-1130 ◽  
Author(s):  
Radhika Gangaraju ◽  
Jihyun Song ◽  
Soo Jin Kim ◽  
Tsewang Tashi ◽  
Brandi N. Reeves ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Hypoxia Inducible Factor ◽  
Chain Reaction ◽  
Thrombosis Risk ◽  
Inflammatory State ◽  
History Of ◽  
Gene Transcripts ◽  
Polymerase Chain

Abstract Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). The pathophysiology of thrombosis in these disorders remains unclear, and we hypothesized that upregulation of thrombotic, inflammatory, and hypoxia-inducible factor (HIF)–regulated genes may play a role in it. We performed unbiased RNA sequencing in granulocytes and platelets of PV patients and found differential expression of several thrombotic, inflammatory, and HIF-regulated genes. The expression of many of these genes positively correlated with JAK2 expression and JAK2V617F allelic burden. We then validated these findings by quantitative polymerase chain reaction analyses of selected gene transcripts in a larger number of PV and ET granulocytes and platelets (58 patients) and in 28 controls, and we compared these findings in patients with and without thrombosis. The study included 29 females and 29 males; of these, 28 had a history of thrombosis. We found that transcripts of several selected genes were upregulated in patients with PV or ET compared with controls. In granulocytes, the expression levels of F3, SELP, VEGFA, and SLC2A1 were significantly higher in patients with a history of thrombosis compared with those who did not have thrombosis. Patients with a history of thrombosis have significantly higher expression of IL1RAP (P &lt; .05) in platelets compared with those without thrombosis. Our study confirms the presence of a thrombo-inflammatory state and augmented HIF activity in PV and ET and its role in thrombosis. These data may provide the background for targeted therapies in PV and ET.

scholarly journals Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages

2019 ◽  
Author(s):  
Xiaoyu Zhao ◽  
Hin Chu ◽  
Bosco Ho-Yin Wong ◽  
Man Chun Chiu ◽  
Dong Wang ◽  
...  
Keyword(s):  
Middle East ◽  
Quantitative Polymerase Chain Reaction ◽  
Severe Disease ◽  
Human Infection ◽  
Middle East Respiratory Syndrome ◽  
Proinflammatory Response ◽  
Pharmacological Inhibition ◽  
Lectin Receptor ◽  
Polymerase Chain

Abstract Background Human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) poses an ongoing threat to public health worldwide. The studies of MERS patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of MERS-CoV. We sought to identify pattern recognition receptor (PRR) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon MERS-CoV infection. Methods The potential signaling pathways were manipulated individually by pharmacological inhibition, small interfering ribonucleic acid (siRNA) depletion, and antibody blocking. The MERS-CoV-induced proinflammatory response was evaluated by measuring the expression levels of key cytokines and/or chemokines. Reverse transcription-quantitative polymerase chain reaction assay, flow cytometry analysis, and Western blotting were applied to evaluate the activation of related PRRs and engagement of adaptors. Results MERS-CoV replication significantly upregulated C-type lectin receptor (CLR) macrophage-inducible Ca2+-dependent lectin receptor (Mincle). The role of Mincle for MERS-CoV-triggered cytokine/chemokine induction was established based on the results of antibody blockage, siRNA depletion of Mincle and its adaptor spleen tyrosine kinase (Syk), and Syk pharmacological inhibition. The cytokine and/or chemokine induction was significantly attenuated by siRNA depletion of retinoic acid-inducible-I-like receptors (RLR) or adaptor, indicating that RLR signaling also contributed to MERS-CoV-induced proinflammatory response. Conclusions The CLR and RLR pathways are activated and contribute to the proinflammatory response in MERS-CoV-infected macrophages.

scholarly journals Possible Role of Trichothecene Mycotoxins in Virulence of Fusarium graminearum on Maize

Plant Disease ◽  
1999 ◽  
Vol 83 (10) ◽  
pp. 954-960 ◽  
Author(s):  
L. J. Harris ◽  
A. E. Desjardins ◽  
R. D. Plattner ◽  
P. Nicholson ◽  
G. Butler ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Fungal Biomass ◽  
Biosynthetic Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Field Trials ◽  
Ear Rot ◽  
Inoculation Method ◽  
Trichodiene Synthase ◽  
Polymerase Chain

Trichothecene-producing and -nonproducing Fusarium graminearum strains were tested for their ability to cause Gibberella ear rot in field trials at two locations—Ottawa, Ontario, and Peoria, Illinois—in 1996. Maize ears were inoculated with wild-type or transgenic F. graminearum strains in which the trichothecene biosynthetic pathway had been disabled by the specific disruption of the trichodiene synthase gene and with a derivative revertant strain in which trichothecene production had been restored through recombination. A silk channel inoculation method was employed at both locations. In addition, a kernel puncture inoculation method was used at the Ontario location. Harvested maize ears were analyzed for visual disease severity, grain yield, deoxynivalenol (DON) concentration, and fungal biomass by quantitative polymerase chain reaction (PCR) and/or ergosterol quantitation. There was a significant correlation (r= 0.86) between data obtained from the two different methods of quantifying fungal biomass. The trichothecene-nonproducing strains were still pathogenic but appeared less virulent on maize than the trichothecene-producing progenitor and revertant strains, as assayed by most parameters. This suggests that the trichothecenes may act as virulence factors to enhance the spread of F. graminearum on maize.

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