Biomedical subjects

Stephen C Robson

Publications and source records attributed to Stephen C Robson.

At least 19 recordsLinked to original sources

PubMed2006

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy.

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.

CD8-Positive T-Lymphocytes↗

PubMed2006

Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy.

Remodeling of uterine spiral arteries is critical for the continuation of a successful pregnancy. Uterine natural killer (uNK) cells are the predominant leukocyte population in the early pregnant decidua, and a role for these cells in spiral artery remodeling in pregnancy has been suggested. Angiogenic growth factors were measured in isolated uNK and total (unseparated) decidual cells (8-10 or 12-14 weeks gestation, n=5 each gestational age) after culture for 48 h. Angiopoietin (Ang)1, placental growth factor, transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF)-C were measured by enzyme-linked immunosorbent assay. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. Levels of Ang2, ICAM-1, and KGF, secreted by the total decidual fraction, decreased with increasing gestational age. uNK levels of Ang2 and VEGF-C also decreased with increasing gestational age. At 8-10 weeks gestation, there was no difference in the level of Ang1, Ang2, TGF-beta1, and VEGF-C secreted by uNK cells and the total decidual fraction. At 12-14 weeks, uNK cells secreted significantly lower levels of VEGF-C than the total decidual fraction. Early pregnancy decidua is a major source of angiogenic growth factors whose levels decrease with increasing gestational age, suggesting that they may play a role in spiral artery remodeling. uNK cells appear to be a prominent source of Ang1, Ang2, TGF-beta1, and VEGF-C within the placental bed.

Angiogenic Proteins↗

PubMed2006

Examining the spatio-temporal expression of mRNA encoding the membrane-bound progesterone receptor-alpha isoform in human cervix and myometrium during pregnancy and labour.

Human parturition is associated with a modification in the sensitivity of the myometrium to progesterone. The molecular basis for this change, however, remains unclear. It is well documented that progesterone can exert its effects through non-genomic mechanisms, including acting through membrane-bound progesterone receptors (mPRs). Recently, a novel membrane-bound PR, termed mPRalpha, was cloned. mPRalpha was unlike any other PR in the databases, but it was seen to have significant homology to G-protein-coupled receptors (GPCR). In this study, we examined the spatio-temporal expression of mPRalpha mRNA in human cervix and both lower and upper myometrial segments from non-pregnant (NP), pregnant (P) and spontaneously labouring (SL) women. We observed an incremental increase in mPRalpha mRNA expression in NP and P samples with the peak level being observed in SL tissues. No major differences were observed between upper or lower pregnant myometrial regions. Interestingly, levels of mPRalpha transcripts were substantially greater in labouring lower segment myometrium compared with labouring upper segment. Significantly, we failed to detect mPRalpha message in either unripe or ripe human cervices. These data suggest that mPRalpha protein function may play a role in regulating lower segment myometrial activity during labour. Whether it functions in the cervix, however, remains unclear.

Adolescent↗

PubMed2006

Interferon-gamma inhibits extravillous trophoblast cell invasion by a mechanism that involves both changes in apoptosis and protease levels.

BACKGROUND: Extravillous trophoblast cell (EVT) invasion of decidua and inner third of the myometrium is critical for a successful pregnancy. Many decidual factors are likely to play a role in regulating this process, including uterine natural killer (uNK) cell-derived cytokines. HYPOTHESES: 1) uNK cells are a major source of IFN gamma (IFN-gamma) and 2) IFN-gamma inhibits EVT invasion via an increase in EVT apoptosis and/or a decrease in active protease levels. METHODS: Total decidual and uNK cells from 8-10 wk and 12-14 wk gestational age were cultured. IFN-gamma mRNA (real-time RT-polymerase chain reaction) and protein levels (FastQuant multicytokine analysis) were determined. EVT invasion in the presence of IFN-gamma or anti-IFN-gamma-neutralizing antibodies was assessed. Trophoblast apoptosis and proliferation was assessed in explants by immunohistochemistry for M30 and Ki67. Substrate zymography was performed to determine levels of secreted MMP2, MMP9, and uPA. RESULTS: mRNA and protein for IFN-gamma was detected in both total decidual and uNK cell fractions. Trophoblast invasion was inhibited by IFN-gamma. The level of M30-positive EVT was increased in the presence of IFN-gamma whereas levels of secreted MMP2 were decreased. CONCLUSIONS: uNK cells are a source of IFN-gamma within early human pregnancy decidua. Mechanisms of IFN-gamma inhibition of EVT invasion include both increased EVT apoptosis and reduced levels of active proteases.

Apoptosis↗

PubMed2006

In vivo analysis of trophoblast cell invasion in the human.

In vivo analysis of trophoblast cell invasion is highly dependent on histological techniques, which are amply described in standard textbooks. The emphasis of this chapter therefore lies on material collection and interpretation of tissue sections, rather than on histological techniques per se. Proper identification of vascular structures on placental bed histological sections is important, the more because invading trophoblastic cells induce significant structural changes in uterine blood vessels, which may be disturbed in complicated pregnancies. Guidelines for distinguishing several vascular structures are provided, and different approaches for qualitative and quantitative assessment of spiral artery changes are discussed. The purpose of such studies is not only to obtain a better insight into mechanisms of trophoblast invasion and associated maternal tissue changes, but also to understand placental bed defects in various pregnancy complications.

Arteries↗

PubMed2005

Low oxygen concentrations inhibit trophoblast cell invasion from early gestation placental explants via alterations in levels of the urokinase plasminogen activator system.

Extravillous trophoblast cell (EVT) invasion in early pregnancy occurs in a relatively low-oxygen environment. The role of oxygen in regulation of EVT invasion remains controversial. We hypothesized that 1) culture in 3% oxygen inhibits EVT invasion compared with culture at 8% or 20% oxygen and 2) inhibition of invasion is due to alterations in levels of components of the urokinase plasminogen activator (PLAU, uPA) system rather than through increased apoptosis and/or decreased proliferation. Placental samples (8-10, 12-14, and 16-20 wk gestation) were obtained from women undergoing elective surgical termination of pregnancy or after cesarean section delivery (term) at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. EVT invasion from placental explants cultured at 3%, 8%, or 20% oxygen was assessed using Matrigel invasion assays. Invasion was assessed on Day 6, explants were harvested for analysis of apoptosis and proliferation, and medium was stored for analysis of PLAU system components by ELISA and casein zymography. Culture at 3% oxygen inhibited EVT invasion. PLAU receptor and plasminogen activator inhibitor-2 protein levels were increased and PLAU activity decreased in these cultures. There was no difference in the proliferation in explants cultured at the three different oxygen concentrations. Apoptosis, assessed by M30 immunostaining, was increased in EVT at both 3% and 8% oxygen. The reduction in the invasive capacity of EVT cultured at 3% oxygen appears to be mediated both by a general inhibition of the PLAU system and a decrease in the number of cells available to invade.

Amyloid beta-Protein Precursor↗

PubMed2005

The switch in alternative splicing of cyclic AMP-response element modulator protein CREM{tau}2{alpha} (activator) to CREM{alpha} (repressor) in human myometrial cells is mediated by SRp40.

The transcription factor cAMP-response element modulator (CREM) protein, plays a major role in cAMP-responsive gene regulation. Biological consequences resulting from the transcriptional stimuli of CREM are dictated by the expression of multiple protein isoforms generated by extensive alternative splicing of its precursor mRNA. We have previously shown that alternative splicing enables the expression of the CREM gene to be "switched" within the human myometrium during pregnancy from the production of CREMtau(2alpha), a potent transcriptional activator to the synthesis of CREMalpha, a transcriptional repressor. Furthermore we have recently reported that this change in the expression of CREM spliced variants is likely to have important ramifications on the regulation of downstream cAMP-response element-responsive target genes involved in uterine activity during gestation. We have investigated the splicing factors involved in controlling the expression of myometrial CREM splice variants. Data presented here from transient transfections indicate that the switch in the synthesis of CREMtau(2)alpha to CREMalpha that occurs during pregnancy is regulated primarily by an SR protein family member, SRp40. We also show that expression of this splicing factor is tightly regulated in the myometrium during pregnancy. SRp40 regulates the splicing of CREM via its interactions with multiple ESE motifs present in the alternatively exons of CREM. In vitro splicing and electrophoretic mobility shift assays were employed to confirm the functionality of the SRp40-binding ESEs, thus providing a mechanistic explanation of how SRp40 regulates the switch in splicing from production of CREMtau(2)alpha to CREMalpha.

Alternative Splicing↗

PubMed2005

Expression of the GTP-binding protein (Galphas) is repressed by the nuclear factor kappaB RelA subunit in human myometrium.

In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor kappaB (NF-kappaB) family of transcription factors. Recently, we have provided evidence that the RelA NF-kappaB subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the Galphas/cAMP/protein kinase A pathway. TNFalpha is a potent activator of NF-kappaB, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNFalpha was observed to repress expression of Galphas while, at the same time, stimulating NF-kappaB activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNFalpha was seen to repress endogenous Galphas mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the Galphas promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of Galphas expression. Together, these data suggest that, in human myometrium, repression of the Galphas gene by NF-kappaB occurs through a non-DNA binding mechanism involving competition for limiting amounts of cellular coactivator proteins including cAMP response element binding protein binding protein.

Cell Line↗

PubMed2005

Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases.

Invasion of extravillous trophoblast cells into the uterus in human pregnancy is tightly regulated. The transforming growth factor-beta (TGFB) family has been suggested to play a role in controlling this process. We hypothesized that TGFB1, 2, and 3 would inhibit the invasive capacity of extravillous trophoblast cells. We also studied trophoblast apoptosis and proliferation and secreted protease levels as potential mechanisms by which these cytokines may act. Inhibition of endogenous TGFB1, 2, and 3 with neutralizing antibodies increased the invasive capacity of extravillous trophoblast cells derived from placental explants. Similarly, addition of exogenous TGFB1, 2, and 3 inhibited the invasive capacity of these cells in a dose-dependent manner. Proliferation of trophoblast in the placental explants did not alter in response to any of the cytokines tested. Apoptosis of villous and extravillous trophoblast did not alter in response to TGFB1, 2, and 3. There was a reduction in secreted levels of matrix metalloproteinase (MMP) 9 and urokinase plasminogen activator in response to all three cytokines. MMP2 and tissue inhibitor of metalloproteinase 1 and 3 levels were not altered. These results suggest that TGFB1, 2, and 3 inhibit trophoblast invasion by a mechanism dependent on reduced protease activity.

Apoptosis↗

PubMed2005

Regulation of expression of the chorionic gonadotropin/luteinizing hormone receptor gene in the human myometrium: involvement of specificity protein-1 (Sp1), Sp3, Sp4, Sp-like proteins, and histone deacetylases.

At present there is little information on the regulatory processes by which the chorionic gonadotropin (CG)/LH receptor gene is regulated in the human myometrium during pregnancy and labor. Employing human primary myometrial cell cultures in conjunction with DNA affinity purification assays/Western analysis, DNA binding studies, CG/LH promoter luciferase reporter gene deletion constructs in transfection assays, and measurement of endogenous mRNA levels in vivo by duplex RT-PCR, we have determined the role that the major transcriptional regulatory sequences from the +1 ATG codon to -2678 bp play in modulating expression of the CG/LH receptor gene in the myometrium. We report that the distal -180 to -2678 bp region of the promoter, although capable of binding members of the Jun family via the multiple activator protein-1 sites within this region, has no significant role in regulating the expression of the CG/LH receptor gene in myometrial cells. In contrast, the two specificity protein-1 to -4 (Sp1-4) GC boxes within the +1 to -180 bp proximal promoter are central to expression of the gene in the myometrium. However, not only are Sp1/Sp3 proteins involved in this process, but Sp4 and a novel Sp-like factor(s) also have an intimate part in transcriptional regulation of the gene. It would appear that Sp1/Sp3/Sp4 and Sp-like proteins are involved in recruiting histone deacetylase complexes to the proximal promoter, preventing chromatin remodeling resulting in transcriptional repression of the gene. Our data suggest that administration of the histone deacetylase inhibitor trichostatin A to human myometrial cells in vitro and in vivo substantially removes this silencing effect on expression of the gene and may implicate the use of this and similar agents in increasing myometrial CG/LH receptor levels and subsequent maintenance of uterine relaxation during fetal maturation.

Base Sequence↗

PubMed2005

Diagnosis and outcome of fetal lower urinary tract obstruction in the northern region of England.

OBJECTIVE: We reviewed the prenatal and postnatal management of fetal lower urinary tract obstruction (LUTO) in a large geographically defined population. METHODS: The records of 113 cases of LUTO seen over a 14-year period were examined. The predictive accuracy of prenatal findings for chronic renal failure (CRF) and a comparison of prenatal-suspected and non-suspected cases were made. RESULTS: The incidence of LUTO was 2.2 in 10 000 births. During the study period, prenatal detection improved from 33 to 62%. Sensitivity of prenatal ultrasound detection of renal dysplasia and fetal urinary sodium, calcium, and beta2-microglobulin for CRF or renal dysplasia on autopsy were 59, 33, 66, and 63% respectively. Compared to undetected cases, those detected prenatally had higher mortality and a higher rate of CRF at 24 months (17% vs 57%, p < 0.01). CONCLUSION: Our observations confirm the poor prognosis associated with fetal LUTO. The value of serial fetal urine biochemistry, other prenatal predictors of postnatal renal function, and the benefits of vesicoamniotic shunting require larger series and longer follow-up.

Adult↗

PubMed2005

Macrophages and not granulocytes are involved in cervical ripening.

To clarify the role of leucocytes in human cervical ripening and dilatation, cervical biopsies were obtained from six non-pregnant women, eight women undergoing early termination of pregnancy, 18 pregnant women undergoing elective Caesarean section at term (both with and without a ripe cervix as determined by Bishop score) and 11 women after term vaginal delivery. Leucocytes were localised by immunohistochemistry labelling and quantified in subepithelial and deep stromal areas. CD45+ leucocytes were more numerous in the subepithelial area of the cervix than in the deep stroma in all groups (P<0.01). CD14+ macrophages and CD15+ granulocytes were increased in both the subepithelial and deep stromal areas only in the vaginal delivery group (P<0.01). The number of macrophages in the ripening cervix (Bishop score above 4) was higher than in the unripe cervix (Bishop score 4 or less; P<0.05) with no differences in other leucocyte populations. CD3+ CD8+ T cells in the subepithelial area were reduced in late pregnancy and after vaginal delivery (P<0.01), but showed no relationship to Bishop score. Macrophages and granulocytes may be involved in the process of cervical dilatation, but macrophage infiltration into the ripening cervix before labour suggests their role in the ripening process. Reduced numbers of CD3+ CD8+ T-lymphocytes in late pregnancy and after vaginal delivery suggests that local immunity is down-regulated in the late pregnancy period. Regional differences in leucocyte subpopulations in the cervix indicate that leucocyte infiltration is likely to be regulated by local factors.

Antigens, CD↗

PubMed2005

Expression and interaction of the transcriptional coregulators, CBP/p300, in the human myometrium during pregnancy and labor.

OBJECTIVE: In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. Recent studies have highlighted a role for the coactivator, CREB binding protein (CBP), in the human myometrium during pregnancy and labor through its ability to acetylate histones. In the present study, the expression of CBP and its related coactivator, p300, were examined. METHODS: Levels and interactions of CBP and its paralogue p300 were determined by Western blotting, immunohistochemistry, and coimmunoprecipitation experiments using myometrial biopsy samples from nonpregnant (NP), pregnant nonlaboring (P), and spontaneously laboring (SL) women. RESULTS: Levels of CBP were seen to increase in term P myometrial samples but were then greatly reduced in SL myometrium. In contrast, levels of p300 remained uniform between NP, P, and SL tissues. These observations were confirmed by immunhistochemical analyses. Immunoprecipitation experiments highlighted that CBP was able to interact with CREB, CREM, ATF-2, and p300 in P lower segment myometrium. CONCLUSION: Recent evidence suggests that competition for CBP plays an important role in regulating gene expression during cell growth. Consequently our data suggest that the increase in myometrial CBP levels during pregnancy may occur to meet this increase in CBP demand. Moreover, from coimmunoprecipitation experiments, this increase in CBP expression would be expected to facilitate the transactivation potential of the cyclic adenosine monophosphate (cAMP)-dependent transcription factors CREB, CREM, and ATF-2.

Adult↗

PubMed2005

Spatial and temporal expression of the myometrial mitogen-activated protein kinases p38 and ERK1/2 in the human uterus during pregnancy and labor.

OBJECTIVE: We have recently identified a novel putative spliced variant of the activating transcription factor 2 (ATF2) in the human myometrium during pregnancy and labor. This protein, termed ATF2-sm like full-length ATF2, acts as a potent transactivator of cyclic adenosine monophosphate response element (CRE)-containing promoter reporter genes. Similarly, employing microarray gene profiling in myometrial cells, we have shown ATF2-sm to affect the expression of several specific myometrial genes associated with regulating uterine activity during pregnancy and labor. At some point after conception this transcription factor becomes spatially expressed within the body of the uterus, with significantly higher levels detected in the upper (corpus) compared to the lower uterine segment. Because ATF2 species are the primary substrate for phosphorylation by the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, the purpose of the current investigation was to define the expression levels of these kinases in upper and lower segment myometrium during pregnancy and labor to see if they also correlated with expression of ATF2-sm. METHODS: Paired myometrial samples were collected from the upper (corpus) and lower uterine segments from term nonlaboring and spontaneously laboring women undergoing elective and emergency cesarean deliveries, respectively. Non-pregnant myometrial samples were collected from premenopausal women having hysterectomies for benign gynecologic disorders. The MAPKs p38 and ERK1/2 present in individual myometrial homogenates were resolved using sodium dodecyl sulfate polacrylamide gel electropheresis (SDS-PAGE) with subsequent Western blotting with specific antibodies and scanning densitometry. Expression of the individual MAPKs in myometrial tissues was confirmed in situ using immunohistochemistry. RESULTS: In non-pregnant tissues, p38 and ERK1/2 expression was uniform throughout the uterus. In term pregnant nonlaboring and spontaneously laboring samples expression of p38 and ERK1 was significantly elevated in the upper uterine segment compared to the lower segment, respectively. In contrast, there was no difference in ERK2 expression. CONCLUSION: The data from this study indicate that both p38 and ERK1 are spatially regulated in different uterine regions during pregnancy/labor and suggest that they may be involved in regulating the activity of ATF2 isoforms and their subsequent effects on myometrial function.

Activating Transcription Factor 2↗

PubMed2005

Identification of human myometrial target genes of the cAMP pathway: the role of cAMP-response element binding (CREB) and modulator (CREMalpha and CREMtau2alpha) proteins.

cAMP-response element (CRE) binding (CREB) and modulator (CREM) proteins, activated by protein kinase A-mediated phosphorylation, bind as homo- and heterodimers to promoters containing CRE and activator protein 1 (AP-1) sites to alter target-gene expression. We have previously reported differential expression of CREB and CREM splice variants CREMalpha and CREMtau2alpha in human myometrium during pregnancy and labour. Via microarray studies with cultured myometrial cells stably transfected with CREB, CREMalpha and CREMtau2alpha cDNAs, CREB affected the expression of 958 genes; 522 being up-regulated and 436 down-regulated. CREMalpha altered the expression of 118 genes; 71 were increased and 47 decreased. CREMtau2alpha affected 220 genes; 148 were activated and 72 repressed. Notably, genes affected by CREB, CREMalpha and CREMtau2alpha belong to largely discrete groups: less than 9% were affected by more than one factor. Genes involved in regulation of cell death and apoptosis, growth and maintenance, signal transduction, physiological and developmental processes, protein kinase cascades, extracellular matrix, cytoskeleton, cell-cycle regulation, transport, and a variety of enzymes, intracellular components and nucleic acid-binding proteins have been described, many of which are involved in the modulation of myometrial activity during pregnancy and parturition.

Cyclic AMP↗

PubMed2004

3 dimensional modelling of early human brain development using optical projection tomography.

BACKGROUND: As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. RESULTS: Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (approximately 300 microm by approximately 150 microm) to the rootlets of cranial nerve XII (approximately 20 microm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is approximately 30 microm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. CONCLUSION: In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of approximately 20 microm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

Brain↗

PubMed2004

Expression and deoxyribonucleic acid-binding activity of the nuclear factor kappaB family in the human myometrium during pregnancy and labor.

In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory factors, many of which are regulated by the nuclear factor kappaB (NF-kappaB) family of transcription factors. The expression and DNA-binding activity of NF-kappaB in the myometrium during gestation and parturition were examined. Levels of c-Rel, p50, and p105 NF-kappaB species were dramatically reduced in pregnant myometrium compared with nonpregnant (NP) controls, whereas expression of the RelA subunit remained uniform. Importantly, during labor, expression of all subunits was observed to be significantly reduced in all myometrial samples studied relative to NP levels. Moreover, for RelA, c-Rel, and p50 subunits, there was a gradient of expression between laboring upper (corpus) and lower uterine segment myometrium. No RelB or p52 subunits could be detected. EMSAs identified changes in NF-kappaB subunit composition in the myometrium during pregnancy and labor, with p50 homodimers predominant in NP tissues being replaced with RelA:p50 heterodimers in pregnant and laboring samples. Significantly, RelA was observed to be phosphorylated at serine-536, implicating the involvement of the phosphatidylinositol-3-kinase/AKT pathway in NF-kappaB function in the myometrium.

Adolescent↗

PubMed2003

Human myometrial quiescence and activation during gestation and parturition involve dramatic changes in expression and activity of particulate type II (RII alpha) protein kinase A holoenzyme.

There are substantial data indicating that components of the cAMP-signaling pathway are differentially expressed in the human myometrium during pregnancy. The effects of cAMP in most tissues and cell types are mainly modulated via protein kinase A, a heterotetrameric protein complex consisting of two regulatory (R) and two catalytic (C) subunits. In the studies presented here, we used specific antibodies in Western blotting/immunoprecipitation, RT-PCR, and functional protein kinase A (PKA) phosphorylation assays to determine the PKA holoenzymes that are expressed in the human myometrium throughout pregnancy and labor. We report that as early as the second trimester of pregnancy, there is a significant increase in expression of the regulatory RII alpha protein subunit of PKA in the myometrium. This increase in protein expression is also mirrored at the mRNA level, indicating transcriptional control throughout pregnancy, whereas during parturition both transcript and protein are significantly decreased. This increase in RII alpha protein also resulted in increased particulate PKA activity in the myometrium during gestation, which was subsequently decreased during labor. Two specific A kinase anchoring proteins, AKAP95 and AKAP79, which have high binding affinities for RII alpha subunits, were found to form complexes with myometrial RII alpha species employing immunoprecipitation assays, but their levels of expression remained uniform in all myometrial tissue samples investigated. Our findings indicate that increased particulate type II PKA activity occurs throughout pregnancy, therefore directing the cAMP quiescence signal to specific subcellular loci within myometrial smooth muscle cells including the contractile machinery at the cytoskeleton; this effect is then removed during parturition.

A Kinase Anchor Proteins↗