Molecular beacons can assess changes in expression and 3′-polyadenylation of human eNOS mRNA

The endothelium plays an essential role in maintaining vascular homeostasis, and it fulfills this role by modulating intracellular signaling and gene expression in response to chemical and mechanical stimuli. Assessing changes in endothelial gene expression is essential to understanding how physiological and pathophysiological processes modulate vascular homeostasis. Here we describe the use of molecular beacons to rapidly and quantitatively assess expression and 3′-polyadenylation of a gene that is important for vascular homeostasis, endothelial nitric oxide synthase (eNOS). Single- and dual-fluorescence resonance energy transfer (FRET) molecular beacon hybridization assays were developed to measure changes in mRNA levels and 3′-polyadenylation, respectively, in primary human endothelial cell cultures subjected to laminar shear stress or statin treatment. Optimized beacon hybridization assays took ∼15 min to perform, and eNOS mRNA levels were validated by quantitative real-time RT-PCR. Competitive inhibition assays and posttranscriptional silencing of eNOS expression were used to verify the specificity of molecular beacon fluorescence. Finally, the dual-FRET method was used to assess eNOS polyadenylation in tissues isolated from mice subjected to exercise training. These data demonstrate that molecular beacons can be used to rapidly and efficiently measure endothelial gene expression and 3′-polyadenylation. This approach could easily be adapted for studies of other endothelial genes and has promise for applications in live endothelial cells.

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Molecular Beacons for Sensitive Gene Detection in Living Cells

Advances in Bioengineering ◽

10.1115/imece2003-42959 ◽

2003 ◽

Author(s):

Andrew Tsurkas ◽

Gang Bao

Keyword(s):

Gene Expression ◽

Real Time ◽

Molecular Beacon ◽

Point Mutations ◽

Living Cells ◽

Molecular Beacons ◽

High Signal ◽

Gene Detection ◽

Real Time Imaging ◽

Background Ratio

Real-time imaging of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of cancer, including cancer diagnosis and analysis. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA as tumor markers due to their high signal-to-background ratio, and their improved specificity in detecting point mutations. However, the harsh intracellular environment does limit the sensitivity of MB-based gene detection. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, or opened due to non-specific interactions. To overcome this difficulty, we have developed a novel dual FRET molecular beacons approach in which a pair of molecular beacons, one with a donor fluorophore and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of false-positive events. We have performed systematic in-solution and cellular studies of dual FRET molecular beacon and demonstrated that this new approach allows for real-time imaging of gene expression in living cells.

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Interactions of Potential Protein Cancer Biomarker Survivin with Plasmonic Nanoparticles and Its Dynamics in Cancer Cells Studied Using Fluorescence Molecular-Beacon Probes, Gated-RET and EQCN Methods

MRS Proceedings ◽

10.1557/opl.2015.137 ◽

2015 ◽

Vol 1720 ◽

Cited By ~ 2

Author(s):

Magdalena Stobiecka ◽

Agata Chalupa ◽

Beata Dworakowska

Keyword(s):

Molecular Beacon ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Fluorescein Isothiocyanate ◽

Cancer Biomarker ◽

Molecular Beacons ◽

Target Sequence ◽

Mrna Levels ◽

Loop Area ◽

Fluorescence Turn On

ABSTRACTThe protein survivin (Sur) has been considered as a potential cancer biomarker due to its involvement in disrupting normal cell cycle by stimulating proliferation and inhibiting cell apoptosis. In this work, we have focused on exploring novel platforms for sensitive monitoring of Sur expression, based on molecular beacons and protein modulation of plasmon-controlled fluorescence. In this framework, we show that Sur can be employed in gating the resonance energy transfer (gRET) between fluorescein isothiocyanate probe dye (FITC) and plasmonic citrate-capped gold nanoparticles (AuNP@Cit). Furthermore, we have designed fluorescent dye-bearing molecular beacons (MBs) targeting nucleotides of the survivin mRNA. The antisense oligonucleotide complementary to the target sequence, inserted in the loop area of the hairpin MB structure, has enabled the fluorescence turn-ON MB switching in the presence of the target, thus signaling the high Sur mRNA levels and enhanced Sur protein expression.

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Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.276.2.l297 ◽

1999 ◽

Vol 276 (2) ◽

pp. L297-L303 ◽

Cited By ~ 45

Author(s):

Robert C. Tyler ◽

Masashi Muramatsu ◽

Steven H. Abman ◽

Thomas J. Stelzner ◽

David M. Rodman ◽

...

Keyword(s):

Gene Expression ◽

Pulmonary Hypertension ◽

Vascular Tone ◽

No Synthase ◽

Enos Gene ◽

No Production ◽

Mrna Levels ◽

Variable Expression ◽

Enos Mrna ◽

Expression And Activity

Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3–4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6–9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 ± 46% of control levels in hypoxic lungs ( P < 0.05) but were decreased by 50 ± 5 and 60 ± 11%, respectively, in monocrotaline-treated and fawn-hooded lungs ( P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 ± 1.4 μM; P < 0.05) but not in either monocrotaline-treated (0.7 ± 0.3 μM) or fawn-hooded (0.45 ± 0.1 μM) lungs vs. normotensive lungs (0.12 ± 0.07 μM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-l-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.

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The Mechanisms Behind the Biological Activity of Flavonoids

Current Medicinal Chemistry ◽

10.2174/0929867325666180706104829 ◽

2019 ◽

Vol 26 (39) ◽

pp. 6976-6990 ◽

Cited By ~ 12

Author(s):

Ana María González-Paramás ◽

Begoña Ayuda-Durán ◽

Sofía Martínez ◽

Susana González-Manzano ◽

Celestino Santos-Buelga

Keyword(s):

Gene Expression ◽

Biological Activity ◽

Phenolic Compounds ◽

Intracellular Signaling ◽

Molecular Mechanisms ◽

Radical Scavenging ◽

Model Organism ◽

Stress Theory ◽

Radical Scavenging Properties

: Flavonoids are phenolic compounds widely distributed in the human diet. Their intake has been associated with a decreased risk of different diseases such as cancer, immune dysfunction or coronary heart disease. However, the knowledge about the mechanisms behind their in vivo activity is limited and still under discussion. For years, their bioactivity was associated with the direct antioxidant and radical scavenging properties of phenolic compounds, but nowadays this assumption is unlikely to explain their putative health effects, or at least to be the only explanation for them. New hypotheses about possible mechanisms have been postulated, including the influence of the interaction of polyphenols and gut microbiota and also the possibility that flavonoids or their metabolites could modify gene expression or act as potential modulators of intracellular signaling cascades. This paper reviews all these topics, from the classical view as antioxidants in the context of the Oxidative Stress theory to the most recent tendencies related with the modulation of redox signaling pathways, modification of gene expression or interactions with the intestinal microbiota. The use of C. elegans as a model organism for the study of the molecular mechanisms involved in biological activity of flavonoids is also discussed.

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Nanocurcumin: A Double-Edged Sword for Microcancers

Current Pharmaceutical Design ◽

10.2174/1381612826666201118100045 ◽

2020 ◽

Vol 26 (45) ◽

pp. 5783-5792

Author(s):

Kholood Abid Janjua ◽

Adeeb Shehzad ◽

Raheem Shahzad ◽

Salman Ul Islam ◽

Mazhar Ul Islam

Keyword(s):

Intracellular Signaling ◽

Dosage Forms ◽

The Body ◽

In Vivo Studies ◽

Mrna Levels ◽

Anticancer Activities ◽

Road Map ◽

Anticancer Effects

There is compelling evidence that drug molecules isolated from natural sources are hindered by low systemic bioavailability, poor absorption, and rapid elimination from the human body. Novel approaches are urgently needed that could enhance the retention time as well as the efficacy of natural products in the body. Among the various adopted approaches to meet this ever-increasing demand, nanoformulations show the most fascinating way of improving the bioavailability of dietary phytochemicals through modifying their pharmacokinetics and pharmacodynamics. Curcumin, a yellowish pigment isolated from dried ground rhizomes of turmeric, exhibits tremendous pharmacological effects, including anticancer activities. Several in vitro and in vivo studies have shown that curcumin mediates anticancer effects through the modulation (upregulation and/or downregulations) of several intracellular signaling pathways both at protein and mRNA levels. Scientists have introduced multiple modern techniques and novel dosage forms for enhancing the delivery, bioavailability, and efficacy of curcumin in the treatment of various malignancies. These novel dosage forms include nanoparticles, liposomes, micelles, phospholipids, and curcumin-encapsulated polymer nanoparticles. Nanocurcumin has shown improved anticancer effects compared to conventional curcumin formulations. This review discusses the underlying molecular mechanism of various nanoformulations of curcumin for the treatment of different cancers. We hope that this study will make a road map for preclinical and clinical investigations of cancer and recommend nano curcumin as a drug of choice for cancer therapy.

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The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4δ2, IL-2, and IFNγ in asthma

Thorax ◽

10.1136/thx.56.7.541 ◽

2001 ◽

Vol 56 (7) ◽

pp. 541-548

Author(s):

E M Glare ◽

M Divjak ◽

M J Bailey ◽

E H Walters

Keyword(s):

Gene Expression ◽

Inhaled Corticosteroids ◽

In Situ Hybridisation ◽

Housekeeping Gene ◽

Cross Sectional Study ◽

Mrna Levels ◽

Competitive Pcr ◽

Cross Sectional ◽

Steroid Use ◽

Biopsy Specimens

BACKGROUNDAsthma has been described as an eosinophilic bronchitis driven by interleukin(IL)-4 and IL-5. The quantification of cytokine mRNA levels in airway samples has been confounded by housekeeping gene expression which differs between and within asthmatics and controls.METHODSThe usefulness of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) that is independent of housekeeping gene expression for quantitating the mRNA for interferon (IFN)γ, IL-2, IL-5, IL-4 and its receptor antagonist encoding splicing variant IL-4δ2 was determined in a cross sectional study of 45 normal control subjects and 111 with asthma.RESULTSAtopic controls and atopic asthmatic subjects expressed more IL-5 than non-atopic controls (p<0.02) in bronchoalveolar lavage (BAL) cells, but not in biopsy specimens. IL-5 mRNA expression in BAL cells from asthmatic subjects using inhaled corticosteroids (ICS) was significantly lower than those not receiving ICS (p=0.04). IL-2 mRNA levels differed with steroid use in biopsy specimens but not in BAL cells. IFNγ, IL-4, and IL-4δ2 mRNA levels did not differ between any groups and were not affected by steroid use. IL-4 and IL-4δ2 mRNA levels were positively correlated (p<0.0001), suggesting coordinated transcription.CONCLUSIONSWhile the signal differentiation of competitive PCR in asthma may rival that of in situ hybridisation and immunohistochemistry, the method is expensive and wasteful of material.

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Methylobacterium oryzae Influences Isoepoxydon Dehydrogenase Gene Expression and Patulin Production by Penicillium expansum

Water ◽

10.3390/w13101427 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1427

Author(s):

Tiago Barros Afonso ◽

Lúcia Chaves Simões ◽

Nelson Lima

Keyword(s):

Gene Expression ◽

Distribution Systems ◽

Water Distribution ◽

Penicillium Expansum ◽

Transcriptional Level ◽

Positive Trend ◽

Mrna Levels ◽

Expression Levels ◽

Production Values ◽

Methylobacterium Oryzae

Biofilms can be considered the main source of microorganisms in drinking water distribution systems (DWDS). The ecology of a biofilm is dependent on a variety of factors, including the presence of microbial metabolites excreted by its inhabitants. This study reports the effect of the Gram-negative bacteria Methylobacterium oryzae on the idh gene expression levels and patulin production of Penicillium expansum mature biofilms. For this purpose, a RT-qPCR method to quantify idh mRNA levels was applied. In addition, the idh expression levels were compared with the patulin production. The results obtained revealed that the effect of the bacterium on pre-established P. expansum biofilms is dependent on the time of interaction. More mature P. expansum biofilms appear to be more resistant to the inhibitory effect that M. oryzae causes towards idh gene expression and patulin production. A positive trend was observed between the idh expression and patulin production values. The results indicate that M. oryzae affects patulin production by acting at the transcriptional level of the idh gene.

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Hepatocellular expression of glucose-6-phosphatase is unaltered during hepatic regeneration

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.4.g717 ◽

1998 ◽

Vol 275 (4) ◽

pp. G717-G722 ◽

Cited By ~ 3

Author(s):

Wisam F. Zakko ◽

Carl L. Berg ◽

John L. Gollan ◽

Richard M. Green

Keyword(s):

Gene Expression ◽

Enzyme Activity ◽

Protein Expression ◽

Partial Hepatectomy ◽

Initial Phase ◽

Physiological Function ◽

Liver Homogenate ◽

Hepatic Regeneration ◽

Mrna Levels ◽

Microsomal Enzyme

Gluconeogenesis and glycogenolysis are essential hepatic functions required for glucose homeostasis. During the initial phase of hepatic regeneration, the immediate-early genes (IEG) are rapidly expressed, and the IEG RL-1 encodes for glucose-6-phosphatase (G-6- Pase). G-6- Pase is a microsomal enzyme essential for gluconeogenesis and glycogenolysis. This study employs a partial-hepatectomy model to examine the expression and activity of G-6- Pase. After partial hepatectomy, rat hepatic G-6- Pase gene expression is transcriptionally regulated, and mRNA levels are increased ≈30-fold. However, in contrast to this rapid gene induction, microsomal enzyme activity is unchanged after partial hepatectomy. Western blotting demonstrates that microsomal G-6- Pase protein expression is also unchanged after partial hepatectomy, and similar results are also noted in whole liver homogenate. Thus, despite marked induction in gene expression of the IEG G-6- Pase after partial hepatectomy, protein expression and enzyme activity remain unchanged. These data indicate that, although this hepatocyte IEG is transcriptionally regulated, the physiologically important level of regulation is posttranscriptional. This highlights the importance of correlating gene expression of IEG with protein expression and physiological function.

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Novel nucleic acid origami structures and conventional molecular beacon–based platforms: a comparison in biosensing applications

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03309-4 ◽

2021 ◽

Author(s):

Noemi Bellassai ◽

Roberta D’Agata ◽

Giuseppe Spoto

Keyword(s):

Nucleic Acid ◽

Structural Properties ◽

Molecular Beacon ◽

Dna Origami ◽

Molecular Beacons ◽

Conformational Polymorphism ◽

Functional Systems ◽

Similarities And Differences ◽

Nucleic Acid Structures ◽

Nucleic Acid Sequences

AbstractNucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Graphical abstract

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Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c946 ◽

1999 ◽

Vol 276 (4) ◽

pp. C946-C954 ◽

Cited By ~ 46

Author(s):

Li Li ◽

Ji Li ◽

Jaladanki N. Rao ◽

Minglin Li ◽

Barbara L. Bass ◽

...

Keyword(s):

Gene Expression ◽

Growth Inhibition ◽

P53 Protein ◽

Specific Inhibitor ◽

P53 Gene ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Polyamine Biosynthesis ◽

Fetal Bovine ◽

P53 Mrna

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

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