16528 Desmet Ct
Spokane Valley WA 99216
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License #: MD60211414
Taxonomy Codes:207X00000X 207XX0005X
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Differential Regulation of Human and Mouse Myometrial Contractile Activity by FSH as a Function of FSH Receptor Density. - Biology of reproduction
Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.Â© 2016 by the Society for the Study of Reproduction, Inc.
The Mre11-Rad50-Xrs2 complex is required for yeast DNA postreplication repair. - PloS one
Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.
Chemotactic activity of gestational tissues through late pregnancy, term labor, and RU486-induced preterm labor in Guinea pigs. - American journal of reproductive immunology (New York, N.Y. : 1989)
Is increased leukocyte chemotactic activity (CA) from gestational tissues necessary for term or preterm labor in guinea pigs?Tissue extracts were prepared from pregnant guinea pig decidua-myometrium, cervix, fetal membranes (amniochorion), and placenta during early third trimester (nÂ =Â 8), term not in labor (TNL, nÂ =Â 5), and term spontaneous labor (TL, nÂ =Â 6), RU486-induced preterm labor (PTL, nÂ =Â 6), or controls (cPTL, nÂ =Â 5). Leukocyte CA was assessed using a modified Boyden chamber assay. Extract chemokine and maternal progesterone concentrations were quantified by enzyme immunoassay.Only the extracts from amniochorion demonstrated increased CA through late gestation and labor. In contrast, CA was decreased in extracts from amniochorion and cervix from animals after RU486-induced PTL. Maternal progesterone concentrations remained high in all groups.Leukocyte CA of intrauterine tissues is increased in term spontaneous labor. However, RU486-induced preterm labor occurs in the absence of increased CA.Â© 2014 The Authors. American Journal of Reproductive Immunology Published by John Wiley & Sons Ltd.
Development and validation of primary human myometrial cell culture models to study pregnancy and labour. - BMC pregnancy and childbirth
The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated.Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; Î± smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1Î² was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni's multiple comparisons test was performed.We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1Î², demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10.Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.
Rho-kinase mediates diphosphorylation of myosin regulatory light chain in cultured uterine, but not vascular smooth muscle cells. - Journal of cellular and molecular medicine
Phosphorylation of myosin regulatory light chain (RLC) triggers contraction in smooth muscle myocytes. Dephosphorylation of phosphorylated RLC (pRLC) is mediated by myosin RLC phosphatase (MLCP), which is negatively regulated by rho-associated kinase (ROK). We have compared basal and stimulated concentrations of pRLC in myocytes from human coronary artery (hVM), which has a tonic contractile pattern to myocytes from human uterus (hUM), which has a phasic contractile pattern. Our studies reveal fundamental differences between hVM and hUM regarding the mechanisms regulating phosphorylation RLC. Whereas hVM responded to stimulation by phosphorylation of RLC at S19, hUM responded by forming diphosphorylated RLC (at T18 and S19; ppRLC), which, compared to pRLC, causes two to threefold greater activation of myosin ATPase that provides energy to power the contraction. Importantly, the conversion of pRLC to ppRLC is mediated by ROK. In hUM, MLCP has high activity for ppRLC and this is inhibited by ROK through phosphorylation of the substrate targeting subunit (MYPT1) at T853. Inhibitors of ROK significantly reduce contractility in both hVM and hUM. We demonstrated that inhibition of ppRLC in phasic myocytes (hUM) is 100-fold more sensitive to ROK inhibitors than is pRLC in tonic myocytes (hVM). We speculate that these differences in phosphorylation of RLC might reflect evolution of different contractile patterns to perform distinct physiological functions. Furthermore, our data suggest that low concentrations of ROK inhibitors might inhibit uterine contractions with minimal effects on vascular tone, thus posing a novel strategy for prevention or treatment of conditions such as preterm birth.Â© 2012 The Authors Journal of Cellular and Molecular Medicine Â© 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
Molecular pathways regulating contractility in rat uterus through late gestation and parturition. - American journal of obstetrics and gynecology
Endogenous uterine agonists can activate numerous signaling pathways to effect increased force. Our objective was to assess expression of key constituents of these pathways, in alliance with contractile function, through late gestation and during term and preterm labor.Using myography, we measured the response to 3 agonists compared with depolarization alone (K(+), 124 mEq/L) and calculated agonist/depolarization ratio. We measured gene expression using quantitative reverse transcription-polymerase chain reaction.Contractile responsiveness to depolarization alone, oxytocin, or endothelin-1 increased during pregnancy compared with nonpregnant animals. The agonist/depolarization ratio did not change during uterine activation or parturition. Inhibition of rhoA-associated kinase decreased responses to oxytocin in all tissues, but significantly more during uterine activation. Expression of rhoA and rhoA-associated kinase was increased significantly in active labor at term or preterm.The rhoA/rhoA-associated kinase pathway is a key regulator of uterine activation during labor and may be a useful target for the prevention of spontaneous preterm birth.Crown Copyright Â© 2012. Published by Mosby, Inc. All rights reserved.
Successful intraoperative retrieval of dislocated femoral trial head during total hip arthroplasty. - The Journal of arthroplasty
Since the advent of modular hip prostheses in the early 1970s, dissociation of the femoral trial head and migration into the deep tissue space of the pelvis are rarely reported complications of total hip arthroplasty. Several case reports have described this complication, but the actual incidence is unknown and likely underreported. Two cases are presented here using a new technique to retrieve a trial femoral head from within the pelvis without use of an extensile approach, laparoscopic retrieval, or secondary incisions.Copyright Â© 2012 Elsevier Inc. All rights reserved.
Association between postoperative Fever and atelectasis in pediatric patients. - World journal for pediatric & congenital heart surgery
Postoperative fever is common after cardiac surgery. In the absence of documented infection, atelectasis is often suggested as a cause of postoperative fever. However, this link is not well supported by pathophysiologic mechanisms. The purpose of this study was to investigate whether an association exists between atelectasis and postoperative fever in pediatric patients undergoing cardiac surgery.A retrospective review was performed on consecutive pediatric patients who underwent cardiac surgery on cardiopulmonary bypass at a single cardiac surgery center from January 1, 2009, to December 31, 2009. Postoperative chest radiographs were evaluated and each lung was scored independently for atelectasis. Clinical parameters including the highest daily recorded temperature were noted and compared to atelectasis data.A total of 203 patients were enrolled; 139 patients (68.5%) had fever at least once during the first 3 postoperative days. The incidence of atelectasis on each day was 41%, 57%, and 71%, respectively. There was no association between fever and atelectasis on any postoperative day (P = .21). Microbiological cultures were performed on 81 patients, and infection was found in 7 patients (3.5%). The frequency of either fever or atelectasis was similar between cyanotic and acyanotic patients.Postoperative fever and atelectasis are both common after pediatric cardiac surgery. In our study, there was no significant association between postoperative fever and atelectasis. In children undergoing cardiac surgery with cardiopulmonary bypass, fever in the postoperative period should not be attributed to atelectasis.
Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes. - PloS one
The 'phosphate-binding tag' (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.
Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots. - PloS one
Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the "in-cell western" (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC(20)) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey(R)) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC(20) phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required.
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16528 Desmet Ct Spokane Valley, WA 99216
1415 N Houk Rd Suite D
1415 N Houk Rd Suite A
12401 E Sinto Ave
420 N Evergreen Rd Bldg B
13103 E Mansfield Incyte Pathology Ps
Spokane, WA 99216