The role of THOA in calcium sensitization in human myometrial smooth muscles
Bibliographic record
Abstract
Improved understanding of the regulation of contractions of uterine smooth muscle (myometrium) is essential to develop more successful strategies for prevention of premature birth, which remains the most common cause of infant death and disability. Oxytocin (OT) and prostaglandin (PG) F2a are potent myometrial stimulants which induce an increase in intracellular Ca++. This activates myosin light chain kinase (MLCK) and subsequently the cell’s contractile machinery. However, there is poor correlation between the rise in Ca++ and the strength of the myometrial contraction. Contractile strength greatly increases at the time of parturition partly due to Ca++-independent factors that sensitize the muscle to the rise in intracellular Ca++. In vascular smooth muscle, one key regulator in this process of Ca++ sensitization is the monomeric G-protein, RhoA which in turn activates Rho-associated kinase (ROK). Studies in the rat myometrium have shown that RhoA and ROK are involved in enhancing OT-induced contractions. The potential role of the RhoA/ROK system in uterine contractility at the time of parturition has not been investigated in the human myometrium or in human cell lines. We propose to investigate the role of RhoA/ROK in the human myometrium using molecular cloning techniques. We have constructed expression plasmids for wild type, dominant positive (constitutively active), and dominant negative (constitutively inactive) isoforms of RhoA and synthesized purified proteins using a bacterial (BL21) translation system. These constructs will be introduced into primary and immortalized human myocytes using the protein transduction domain (TAT) derived from the HIV virus, which is capable of introducing whole proteins into mammalian cells. RhoA activation/translocation to the plasma membrane will be visualized using real time confocal microscopy in experiments where the RhoA proteins have been tagged with green fluorescent protein (GFP). Following introduction of the normal and mutant G-proteins, the downstream targets of activated ROK will be assayed for phosphorylation status using near infrared (NIR) fluorescence imaging of western blots or in-cell westerns. These targets include the myosin binding subunit (MBS) of myosin light chain phosphatase (MLCP), and two peptide phosphatase inhibitors of MLCP, CPI-17 and PHI-1. In addition, we propose to measure ROK activity directly using a direct enzyme assay. We will monitor calcium transients using fluorescence microscopy to verify the calcium independence of our measurements. These experiments will determine the role of the RhoA/ROK system in the mechanisms that may determine human uterine contractility. This information may direct new strategies to prevent or treat preterm labour.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".