scholarly journals Praziquantel Affects the Regulatory Myosin Light Chain of Schistosoma mansoni

2008 ◽  
Vol 53 (3) ◽  
pp. 1054-1060 ◽  
Author(s):  
Munirathinam Gnanasekar ◽  
Ashok M. Salunkhe ◽  
A. Krishna Mallia ◽  
Yi Xun He ◽  
Ramaswamy Kalyanasundaram
Keyword(s):  
Schistosoma Mansoni ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Dna Analysis ◽  
Peptide Mapping ◽  
Immunoblot Analysis ◽  
Drug Of Choice ◽  
Regulatory Myosin Light Chain

ABSTRACT Praziquantel (PZQ) is the drug of choice for schistosomiasis and probably is the only highly effective drug currently available for treating schistosomiasis-infected individuals. The mode of action of PZQ involves increasing the calcium uptake of the parasite, resulting in tegumental damage and death of the parasite. Despite its remarkable function, the target of PZQ has not been identified yet. To begin to understand where PZQ acts, in this study we expressed the cDNA library of Schistosoma mansoni on the surface of T7 bacteriophages and screened this library with labeled PZQ. This procedure identified a clone that strongly bound to PZQ. Subsequent DNA analysis of inserts showed that the clone coded for regulatory myosin light chain protein. The gene was then cloned, and recombinant S. mansoni myosin light chain (SmMLC) was expressed. Immunoblot analysis using antibodies raised to recombinant SmMLC (rSmMLC) showed that SmMLC is abundantly expressed in schistosomula and adult stages compared to the amount in cercarial stages. In vitro analyses also confirmed that PZQ strongly binds to rSmMLC. Further, peptide mapping studies showed that PZQ binds to amino acids 46 to 76 of SmMLC. Immunoprecipitation analysis confirmed that SmMLC is phosphorylated in vivo upon exposure to PZQ. Interestingly, significant levels of anti-SmMLC antibodies were present in vaccinated mice compared to the amount in infected mice, suggesting that SmMLC may be a potential target for protective immunity in schistosomiasis. These findings suggest that PZQ affects SmMLC function, and this may have a role in PZQ action.

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

scholarly journals Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

2009 ◽  
Vol 297 (6) ◽  
pp. H2242-H2252 ◽  
Author(s):  
Ravi Goyal ◽  
Ashwani Mittal ◽  
Nina Chu ◽  
Lijun Shi ◽  
Lubo Zhang ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Cerebral Arteries ◽  
Age Groups ◽  
Rho Kinase ◽  
Immunoblot Analysis ◽  
Mek Inhibitor ◽  
Inhibitory Protein ◽  
Regulatory Myosin Light Chain

Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

Nerve-dependent accumulation of myosin light chain 3 in developing limb musculature

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 673-684
Author(s):  
P.A. Merrifield ◽  
I.R. Konigsberg
Keyword(s):  
Neural Tube ◽  
Light Chain ◽  
Limb Development ◽  
Myosin Light Chain ◽  
Chorioallantoic Membrane ◽  
In Ovo ◽  
Fast Myosin ◽  
Limb Musculature

Myosin alkali light chain accumulation in developing quail limb musculature has been analysed on immunoblots using a monoclonal antibody which recognizes an epitope common to fast myosin light chain 1 (MLC1f) and fast myosin light chain 3 (MLC3f). The limb muscle of early embryos (i.e. up to day 10 in ovo) has a MLC profile similar to that observed in myotubes cultured in vitro; although MLC1f is abundant, MLC3f cannot be detected. MLC3f is first detected in 11-day embryos. To determine whether this alteration in MLC3f accumulation is nerve or hormone dependent, limb buds with and without neural tube were cultured as grafts on the chorioallantoic membrane of chick hosts. Although differentiated muscle develops in both aneural and innervated grafts, innervated grafts contain approximately three times as much myosin as aneural grafts. More significantly, although aneural grafts reproducibly accumulate normal levels of MLC1f, they fail to accumulate detectable levels of MLC3f. In contrast, innervated grafts accumulate both MLC1f and MLC3f, suggesting that the presence of neural tube in the graft promotes the maturation, as well as the growth, of muscle tissue. This is the first positive demonstration that innervation is necessary for the accumulation of MLC3f that occurs during normal limb development in vivo.

Substance P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase

1990 ◽  
Vol 259 (2) ◽  
pp. C258-C265 ◽  
Author(s):  
M. A. Corson ◽  
J. R. Sellers ◽  
R. S. Adelstein ◽  
M. Schoenberg
Keyword(s):  
Substance P ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Time Course ◽  
Myosin Light Chain ◽  
Immunoblot Analysis ◽  
Isometric Tension ◽  
Peak Tension ◽  
Tension Response

At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.

Sign in / Sign up

Export Citation Format

Share Document