scholarly journals Salvia sclarea L. Essential Oil Extract and Its Antioxidative Phytochemical Sclareol Inhibit Oxytocin-Induced Uterine Hypercontraction Dysmenorrhea Model by Inhibiting the Ca2+–MLCK–MLC20 Signaling Cascade: An Ex Vivo and In Vivo Study

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 991
Author(s):  
Jennifer Wong ◽  
Yi-Fen Chiang ◽  
Yin-Hwa Shih ◽  
Chun-Hui Chiu ◽  
Hsin-Yuan Chen ◽  
...  
Keyword(s):  
Essential Oil ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Uterine Contraction ◽  
Ex Vivo ◽  
In Vivo Study ◽  
Writhing Test ◽  
Salvia Sclarea ◽  
Sd Rats

Salvia sclarea essential oil is used as an aromatic therapy for dysmenorrhea. Sclareol—one of the natural products isolated from S. sclarea—displays anti-inflammatory and antioxidant activities; however, researchers have not yet evaluated the mechanism related to the pain-relieving effect of sclareol. In the present study, we aimed to investigate the potential effect of sclareol in ex vivo and in vivo dysmenorrhea models, as well as its possible mechanism. In the ex vivo study of uterine tissue from Sprague Dawley (SD) rats, the uterine contraction amplitude was observed and recorded. In the in vivo study, we measured the uterine contraction pressure of SD rats and performed writhing tests on mice. The uterine tissues from the writhing test subjects were collected and analyzed by Western blot. The results demonstrated that sclareol inhibited prostaglandin (PG) F2α-, oxytocin-, acetylcholine-, carbachol-, KCl-, and Bay K 8644-induced uterine contraction and possessed an analgesic effect in the writhing test. Sclareol affects the Ca2+ level and regulates oxytocin receptor (OTR), myosin light chain kinase (MLCK), extracellular signal-regulated kinase, p-p38, cyclooxygenase-2 (COX-2), and phospho-myosin light chain 20 (p-MLC20) protein expression. Integrating these results, we suggest that sclareol is a potential alternative supplement for dysmenorrhea.

scholarly journals Ethanolic Extracts of Adlay Testa and Hull and Their Active Biomolecules Exert Relaxing Effect on Uterine Muscle Contraction through Blocking Extracellular Calcium Influx in Ex Vivo and In Vivo Studies

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 887
Author(s):  
Yun-Ju Huang ◽  
Yu-Chieh Chen ◽  
Hsin-Yuan Chen ◽  
Yi-Fen Chiang ◽  
Mohamed Ali ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Uterine Contraction ◽  
Calcium Influx ◽  
Ex Vivo ◽  
Ethanolic Extract ◽  
Ethyl Acetate Fraction ◽  
In Vivo Studies ◽  
Human Myometrium ◽  
Writhing Test

Dysmenorrhea is one of the most prevalent disorders in gynecology. Historically, adlay (Coix lachryma-jobi L. var. Ma-yuen Stapf.) has been explored for its anti-tumor, pain relief, anti-inflammatory, and analgesic effects. The aim of this study was to evaluate the effects of adlay seeds on the inhibition of uterine contraction and thus dysmenorrhea relief, in vitro and in vivo. HPLC-MS and GC were used to elucidate the ethyl acetate fraction of adlay testa ethanolic extract (ATE-EA) and ethyl acetate fraction of adlay hull ethanolic extract (AHE-EA). Elucidation yielded flavonoids, phytosterols, and fatty acids. Uterine leiomyomas and normal adjacent myometrial tissue were evaluated by oxytocin- and PG-induced uterine contractility. ATE-EA and AHE-EA suppressed uterine contraction induced by prostaglandin F2 alpha (PGF2α), oxytocin, carbachol, and high-KCl solution ex vivo. In addition, the external calcium (Ca2+) influx induced contraction, and increased Ca2+ concentration was inhibited by ATE-EA and AHE-EA on the uterine smooth muscle of rats. Furthermore, ATE-EA and AHE-EA effectively attenuated the contraction of normal human myometrium tissues more than adjacent uterine leiomyoma in response to PGF2α. 3,5,6,7,8,3′,4′-Heptamethoxyflavone and chrysoeriol produced a remarkable inhibition with values of IC50 = 24.91 and 25.59 µM, respectively. The experimental results showed that treatment with ATE-EA at 30 mg/day effectively decreased the writhing frequency both on the oxytocin-induced writhing test and acetic acid writhing test of the ICR mouse.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Srikanth Perike ◽  
Frederick Damen ◽  
Andrielle Capote ◽  
Katherina M Alsina ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates

1985 ◽  
Vol 5 (11) ◽  
pp. 3168-3182
Author(s):  
E E Strehler ◽  
M Periasamy ◽  
M A Strehler-Page ◽  
B Nadal-Ginard
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Mammalian Species ◽  
In Vitro Transcription ◽  
Chain Gene ◽  
Promoter Regions ◽  
Light Chain Gene ◽  
Direct Initiation

DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence. The above findings indicate that the overall structure of the MLC1f/3f genes has been maintained between avian and mammalian species and that these genes contain two functional and widely spaced promoters. The fact that the structures of the alkali light chain gene from Drosophila melanogaster and of other related genes of the troponin C supergene family resemble a MLC3f gene without an upstream promoter and first exon strongly suggests that the present-day MLC1f/3f genes of higher vertebrates arose from a primordial alkali light chain gene through the addition of a far-upstream MLC1f-specific promoter and first exon. The two promoters have evolved at different rates, with the MLC1f promoter being more conserved than the MLC3f promoter. This discrepant evolutionary rate might reflect different mechanisms of promoter activation for the transcription of MLC1f and MLC3f RNA.

The Regulation of Myosin Light Chain Kinase by Ca++ and Calmodulin in Vitro and in Vivo

1982 ◽  
pp. 219-238 ◽  
Author(s):  
J. T. Stull ◽  
D. K. Blumenthal ◽  
B. R. Botterman ◽  
G. A. Klug ◽  
D. R. Manning ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain

Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human Uterine Myocytes

2018 ◽  
Vol 26 (7) ◽  
pp. 988-996
Author(s):  
Bryan F. Mitchell ◽  
Mei Chi ◽  
Elle Surgent ◽  
Bailey M. Sorochan ◽  
Curtis N. Tracey ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Ex Vivo ◽  
Neonatal Morbidity ◽  
The Novel ◽  
Uterine Contractility ◽  
Uterine Activity ◽  
Kinase C

Background: Preterm birth is the most common cause of neonatal morbidity and mortality and a common precedent to lifelong disability. Current treatment has minimal efficacy. Objective: We assessed the role of isozymes of the protein kinase C (PKC) family in regulating the phosphorylation of myosin regulatory light chains (RLCs), which regulate uterine contractility. We also explored the mechanisms through which these isozymes function. Study Design: We used a previously characterized and validated quantitative in-cell Western (ICW) assay to measure site-specific phosphorylations on myosin RLC and CPI-17. Cultures of human uterine myocytes (hUM) were treated with the potent contractile stimulant oxytocin to induce uterine contractility or a pharmacological mimic of diacyl-glycerol to stimulate the conventional and novel isozymes of the PKC family. Combinations of isozyme-selective inhibitors were used to determine the effects of the conventional and novel classes of isozymes. Results: Stimulation of PKC using phospho-dibutyrate caused immediate, concentration-dependent inhibition of uterine activity ex vivo. Using the ICW assay with hUM, the oxytocin-stimulated increase in the pro-contractile phosphorylations of myosin RLCs at serine19 and threonine18 was completely inhibited by prior treatment with phorbol-12-myristate-13-acetate, which stimulates both convention and novel classes of isozymes. Our results suggest that the conventional class of isozymes cause a reduction in phosphorylations at serine19 and threonine18 by reducing activity of myosin light chain kinase. The novel class of isozymes has 2 mechanisms of action: the first is activation of CPI-17 through phosphorylation at threonine38, which results in reduced activity of myosin light chain phosphatase and increased levels of activated myosin RLC; the second is increased phosphorylation of the N-terminal region of myosin RLC. Conclusions: Specific agonists for the conventional isozymes or inhibitors of the novel isozymes of the PKC family could be useful pharmacological agents for regulation of uterine activity.

scholarly journals Fibroblast Protection of Borrelia burgdorferi from Doxycycline, Cefuroxime and Daptomycin Combination is Eliminated by Oregano or Carvacrol Essential Oil

2019 ◽  
Author(s):  
Chunxiang Bai ◽  
Hua Yang ◽  
Peng Cui ◽  
Rong Quan ◽  
Ying Zhang
Keyword(s):  
Lyme Disease ◽  
Essential Oil ◽  
Borrelia Burgdorferi ◽  
Drug Combination ◽  
Defense Mechanisms ◽  
Ex Vivo ◽  
Fibroblast Cells ◽  
Murine Fibroblast ◽  
Drug Regimens

AbstractBorrelia burgdorferi could be occasionally recovered from patients after antibiotic treatment, which indicates it may resist eradication by antibiotic and host defense mechanisms. Skin fibroblast cells have previously been shown to protect the killing of B. burgdorferi by ceftriaxone, a powerful antibiotic commonly used to treat Lyme disease. In this study, we evaluated if fibroblast cells could also protect against the doxycycline+ cefuroxime+ daptomycin drug combination which has previously been shown to completely eradicate highly persistent biofilm-like microcolonies of B. burgdorferi. To do so, we utilized a GFP-labeled B. burgdorferi for infection of murine fibroblast cells and assessed the effect of the drug combination on killing the bacteria in the presence or absence of the fibroblast cells. Surprisingly, we found that fibroblasts could protect B. burgdorferi from being completely killed by the drug combination doxycycline, cefuroxime and daptomycin, which eradicated B. burgdorferi completely in the absence of fibroblast cells. Interestingly, addition of essential oil carvacrol or oregano at 0.1% could enhance the activity of the doxycycline+ cefuroxime+ daptomycin drug combination and led to complete eradication of B. burgdorferi even in the presence of fibroblast cells. Further studies are needed to determine if the essential oil drug combinations could eradicate persistent B. burgdorferi infection in vivo in animal models. Our study provides a useful and convenient ex vivo model for evaluating different drug regimens needed for developing more effective treatment of persistent Lyme disease in the future.

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