Real-Time Polymerase Chain Reaction to Quantify mRNA for Endothelial Nitric Oxide Synthase

2004 ◽  
pp. 125-132
Author(s):  
Yi Chu ◽  
Frank M. Faraci
Keyword(s):  
Nitric Oxide ◽  
Polymerase Chain Reaction ◽  
Nitric Oxide Synthase ◽  
Real Time ◽  
Endothelial Nitric Oxide Synthase ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Hypothalamic nitric oxide synthase is depressed in cholestatic rats

1997 ◽  
Vol 272 (5) ◽  
pp. G1034-G1040 ◽  
Author(s):  
M. G. Swain ◽  
T. Le ◽  
A. W. Tigley ◽  
P. Beck
Keyword(s):  
Nitric Oxide ◽  
Polymerase Chain Reaction ◽  
Steady State ◽  
Nitric Oxide Synthase ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Bile Duct Resection ◽  
Protein Levels ◽  
Polymerase Chain ◽  
Oxide Synthase

We examined hypothalamic nitric oxide synthase (NOS) levels and release as well as steady-state mRNA levels in rats with cholestasis due to bile duct resection (BDR) and in sham-resected control rats. BDR rats had a significant reduction in hypothalamic NOS-containing neurons in the hypothalamic paraventricular nucleus as determined by NADPH-diaphorase staining, compared with sham-resected controls. In addition, NOS activity, measured indirectly by determining nitrite release from hypothalamic explants, was significantly lower in BDR rats compared with sham-resected animals. Hypothalamic steady-state NOS mRNA levels [brain constitutive NOS (bNOS)] were determined by semiquantitative reverse transcription-polymerase chain reaction and were found to be increased 1.5-fold in BDR rats compared with sham rats. In summary, BDR rats have diminished hypothalamic NOS levels and activity coupled with enhanced steady-state bNOS mRNA levels, suggesting that depressed hypothalamic NOS protein levels are due to posttranscriptional defects.

scholarly journals Endothelial Nitric Oxide Synthase Knockdown in Human Stem Cells Impacts Mitochondrial Biogenesis and Adipogenesis: Live-Cell Real-Time Fluorescence Imaging

2021 ◽  
Vol 10 (4) ◽  
pp. 631
Author(s):  
Sylvia Lee-Huang ◽  
Philip Lin Huang ◽  
Paul Lee Huang
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Stem Cell ◽  
Nitric Oxide Synthase ◽  
Real Time ◽  
Mitochondrial Biogenesis ◽  
Endothelial Nitric Oxide Synthase ◽  
Live Cell ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

Effect of NOS Inhibitor on Cytokine and COX2 Expression in Rat Pulpitis

2005 ◽  
Vol 84 (8) ◽  
pp. 762-767 ◽  
Author(s):  
N. Kawashima ◽  
H. Nakano-Kawanishi ◽  
N. Suzuki ◽  
M. Takagi ◽  
H. Suda
Keyword(s):  
Nitric Oxide ◽  
Polymerase Chain Reaction ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Reverse Transcription ◽  
Chain Reaction ◽  
Nos Inhibitor ◽  
Polymerase Chain ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Various kinds of chemical mediators are synthesized in the course of pulpitis; thus, control of their production would assist in inducing a reduction in pulpal inflammation. We hypothesized that nitric oxide (NO) would be an important mediator of pulpal inflammation. Pulpal inflammation was induced by the application of LPS in rat incisor pulp, and inducible nitric oxide synthase (iNOS) expression was evaluated by reverse-transcription/polymerase chain-reaction and immunohistochemical staining. After LPS application, iNOS mRNA was first detected after 3 hrs, peaked at 6 hrs, and decreased thereafter. iNOS-positive cells were macrophages and neutrophils. An NOS inhibitor caused drastic decreases in the expression of pro-inflammatory cytokines and COX2 mRNA, which was highly induced in the LPS-induced pulpitis. These results indicate that NO synthesis is related to the initiation of mediator production, and that its down-regulation should contribute to the prevention of pro-inflammatory mediator synthesis. Abbreviations: ANOVA, analysis of variance; COX2, cyclo-oxygenase 2; EDTA, ethylenediaminetetraacetic acid; iNOS, inducible nitric oxide synthase; IL, interleukin; L-NAME, NG-nitro L-arginine methyl ester; LPS, lipopolysaccharide; NO, nitric oxide; NOS, nitric oxide synthases; PG, prostaglandin; RT-PCR, reverse-transcription/polymerase chain-reaction; TNFα, tumor necrosis factor alpha.

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