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G D Bryant-Greenwood

Publications and source records attributed to G D Bryant-Greenwood.

At least 19 recordsLinked to original sources
PubMed2006

Pre-B-cell colony-enhancing factor (PBEF/Visfatin) gene expression is modulated by NF-kappaB and AP-1 in human amniotic epithelial cells.

A localized intrauterine inflammatory response is often associated with the initiation of normal human parturition, whereas infection causes a similar but more florid response initiating preterm labor. Pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes and is up-regulated by labor, severe infection and inflammatory stimuli. The aim of this study was to determine the involvement of the transcription factors NF-kappaB and AP-1 in the response of PBEF to an inflammatory stimulus and compare it with IL-8. The results showed that this treatment of amniotic epithelial-like cells (WISH) and primary amniotic epithelial cells increased expression of PBEF and IL-8, but IL-8 responded 100-fold more than PBEF. IL-1beta treatment together with a panel of NF-kappaB and AP-1 inhibitors demonstrated the involvement of these transcription factors in the up-regulation of PBEF. These data show that an inflammatory stimulus in the fetal membranes inducing NF-kappaB and AP-1 would up-regulate PBEF as well as IL-8.

Amnion↗
PubMed2005

The human relaxin receptor (LGR7): expression in the fetal membranes and placenta.

The relaxin receptor has been recently described as a leucine-rich repeat G-protein coupled receptor and designated as LGR7. A closely related receptor, LGR8, is co-expressed by some cells. This study explored the expression of the genes for these receptors in the human fetal membranes and placenta by RT-PCR and the LGR7 protein by immunolocalization. The results showed that LGR7 was well expressed in the fetal membranes, with significantly more in the decidua (p<0.05) than in the amnion. On the other hand, relatively low levels were expressed in the placenta. The major splice variant of LGR7 was undetectable in either the placenta or fetal membranes. Expression of LGR8 was also below the level of detectability in either tissue. Immunostaining for LGR7 was conducted with antisera to both its endodomain and ectodomain, in order to seek evidence for a solubilized ectodomain. However, similar staining patterns were obtained with both antisera, with predominant staining in the cells of the amniotic epithelium, chorionic cytotrophoblast and decidua. Full-thickness fetal membranes from preterm deliveries, before and after labor or after preterm premature rupture of the membrane (PPROM) and labor were collected. In addition, membranes at term, both before and after spontaneous labor were used for analysis of LGR7 gene expression. There was significantly greater LGR7 expressed (p=0.01) in the preterm period compared to term, indicating a potentially important role for relaxin at this time. There was a marginal decline in LGR7 gene expression after labor and delivery both at preterm and term, which did not reach significance. Immunostaining patterns showed less inter-patient variability than did gene expression, with more intense staining for LGR7 after labor and delivery.

Adult↗
PubMed2001

Lysyl oxidases: expression in the fetal membranes and placenta.

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions. Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.

Abortion, Legal↗
PubMed2001

Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes.

Pre-B-cell colony enhancing factor (PBEF) was first isolated from an activated peripheral blood lymphocyte cDNA library and was found to be involved in the maturation of B-cell precursors. It was subsequently identified as one of the genes upregulated by distending the human fetal membranes in vitro. Here we report on the genomic organization of this gene, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. Neither the gene nor the protein has any homology with other cytokines in any currently available database. The use of two promoters (proximal and distal) may result in differential, tissue specific expression of the PBEF transcripts. The 5'-flanking region lacks the classical sequence motif that would place it with the hematopoietic cytokines; however, it has several putative regulatory elements, suggesting that this gene may be chemically and mechanically responsive to inducers of transcription. The three PBEF mRNA transcripts were observed in both normal and infected human fetal membranes but were significantly upregulated (P<0.05) in severe infection. The PBEF protein was immunolocalized, in both normal and infected tissues, to both the normal fetal cells of the amnion and chorion and the maternal decidua of the membranes, and to the invading neutrophils. These stained strongly and were likely to contribute to the increased expression in infection. The amniotic epithelial cell line (WISH cells) has been used as a model to study PBEF gene modulation. Lipopolysaccharide, interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha and IL-6 all significantly increased the expression of PBEF in 4 h of treatment. The addition of dexamethasone to IL-1beta and TNFalpha significantly reduced the response of PBEF to these cytokines. IL-8 treatment failed to alter PBEF gene expression. Thus PBEF is a cytokine expressed in the normal fetal membranes and upregulated when they are infected. It is likely to have a central role in the mechanism of infection-induced preterm birth.

Blotting, Northern↗
PubMed2000

Fetal membrane distention: I. Differentially expressed genes regulated by acute distention in amniotic epithelial (WISH) cells.

OBJECTIVE: This study was undertaken to determine which genes were up-regulated by acute distention in an amniotic epithelial cell line and in human fetal membranes. STUDY DESIGN: WISH cells, a human amniotic epithelial cell line, were grown on silicone elastomer sheets coated with extracellular matrix and reproducibly distended by 40% in a novel device for 4 hours. Differential gene expression was analyzed by means of suppression subtractive hybridization. Expression of the identified genes was then quantitated by Northern blot analysis in fetal membrane explants after distention in the same device for 4 hours. The effect of distention on apoptosis of the cells and tissue samples was concomitantly studied by means of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. RESULTS: The genes for interleukin 8 and pre-B-cell colony-enhancing factor were found to be up-regulated in both the WISH cells and the distended fetal membranes. The apoptotic index values in both the cells and the tissue samples were unaffected by distention. CONCLUSIONS: Acute distention induces the up-regulation of interleukin 8 and pre-B-cell colony-enhancing factor in both WISH cells and human fetal membranes and does not cause apoptosis.

Amnion↗
PubMed2000

Human fetal membranes: their preterm premature rupture.

At the 1999 annual meeting of the Society for the Study of Reproduction there were three speakers in the minisymposium entitled "I've got to get out of here: fetal-maternal interactions involved in parturition". The primary focus was on research progress in understanding the mechanisms involved in human parturition. Although the title of the symposium emphasized the need to "get out", there was considerable emphasis on understanding the problem of "getting out too early" or preterm birth. While preterm birth is unusual in most species, it is of major clinical importance in the human. The data presented by one of the speakers is reviewed here with a focus on preterm labor and preterm premature rupture of the fetal membranes as mechanisms involved in the diverse pathology of preterm birth.

Adult↗
PubMed2000

Isolation and analysis of the 3'-untranslated regions of the human relaxin H1 and H2 genes.

The human has two relaxins, termed H1 and H2, both of which are biologically active and co-expressed in the decidua, placenta and prostate; in the corpus luteum, the main source of circulating relaxin, only the H2 form is expressed. The reasons for this differential expression of the relaxin genes are unknown. The possibility that their 3'-untranslated regions (UTRs) contribute to this differential expression by affecting their mRNA stabilities was investigated. Thus the 3'-UTRs of both relaxin genes were isolated through a combined 3'-rapid amplification of cDNA ends-PCR (RACE-PCR) using poly (A)(+)RNA from human decidua, placenta, prostate and corpus luteum. The sequences obtained for each 3'-UTR were identical in the tissues examined, were AT-rich (72%) and showed 91% homology between relaxin H1 and H2 when maximally aligned to include several gaps, the significance of which is unknown. Relaxin H1 has two, and relaxin H2 has one, poly (A)(+) signal, in addition to one cytoplasmic polyadenylation element 30 nucleotides upstream of this. The mRNA levels of relaxin H1 and H2 in the prostate adenocarcinoma LNCaP.FGC cell line were determined by quantitative competitive RT-PCR. Relaxin H1 had a 10-fold greater number of molecules (approximately 2.5x10(7)) per microgram of total RNA than relaxin H2 (approximately 2.5x10(6)). The stability of relaxin H1 and H2 mRNAs were compared in LNCaP cells treated with the transcription inhibitor actinomycin D (10 mM) for 0, 1, 2, 4, 8, 10, 14, or 24 h. Half-lives of 3.17 days for relaxin H1 mRNA and 11. 4 h for relaxin H2 mRNA were obtained from semi-logarithmic plots. Thus both mRNAs are relatively stable; however, relaxin H1 mRNA is considerably more stable than relaxin H2, at least in LNCaP cells. This difference in their mRNA stability may partly explain the greater level of expression of relaxin H1 in these cells.

3' Untranslated Regions↗
PubMed1999

The LOXL2 gene encodes a new lysyl oxidase-like protein and is expressed at high levels in reproductive tissues.

We have reported in this paper the complete cDNA sequence, gene structure, and tissue-specific expression of LOXL2, a new amine oxidase and a member of an emerging family of human lysyl oxidases. The predicted amino acid sequence, from several overlapping cDNA clones isolated from placenta and spleen cDNA libraries, shared extensive sequence homology with the conserved copper-binding and catalytic domains of both lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) protein. These conserved domains are encoded by five consecutive exons within the LOX, LOXL, and LOXL2 genes that also maintained exon-intron structure conservation. In contrast, six exons encoding the amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is present in LOX and LOXL, suggesting a different processing and intracellular localization for this new protein. Expression of the LOXL2 gene was detected in almost all tissues with the highest steady state mRNA levels in the reproductive tissues, placenta, uterus and prostate. In situ hybridization identified placental syncytial and cytotrophoblasts responsible for the synthesis of LOXL2 mRNA and demonstrated a spatial and temporal expression pattern unique to the LOXL2 gene.

Amino Acid Oxidoreductases↗
PubMed1999

Genes upregulated in human fetal membranes by infection or labor.

OBJECTIVE: To determine whether suppression subtractive hybridization can detect genes in fetal membranes that are upregulated by infection, preterm premature rupture of membranes (PROM), or labor. METHODS: Using suppression subtractive hybridization, messenger RNAs from a preterm fetal membrane obtained at cesarean delivery without labor (control) were subtracted from a pool of messenger RNAs of three patients with preterm PROM and vaginal delivery. Eight candidate genes identified as upregulated were quantitated by Northern analysis in each of the tissues and in additional patient subgroups. RESULTS: Eight differentially upregulated genes were identified in preterm labor with PROM. Four of the genes are known to be involved in the response to inflammation or infection, and subsequent histologic examination showed one of the preterm PROM tissues to be infected. F-actin capping protein and chitinase precursor, not previously known to be involved in infection, were also upregulated in the infected tissue from preterm PROM. Northern blots using additional subgroups of patients showed that a regulatory G-protein signaling protein gene was significantly upregulated at term by labor in addition to significant upregulation of interleukin-8. There was a strong correlation between the gene expression for complement factor-B and duration of membrane rupture in the patients with preterm PROM. CONCLUSION: Two novel genes potentially involved in the response to inflammation or infection have been identified. A regulatory G-protein signaling protein and interleukin-8 gene expression were upregulated by labor. Complement factor-B gene expression was directly related to the duration of membrane rupture.

Adult↗
PubMed1999

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term.

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.

Amnion↗
PubMed1999

Analysis of the 5'-upstream regions of the human relaxin H1 and H2 genes and their chromosomal localization on chromosome 9p24.1 by radiation hybrid and breakpoint mapping.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

5' Untranslated Regions↗
PubMed1998

A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection.

OBJECTIVE: This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis. STUDY DESIGN: Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes (n = 17) or preterm labor (n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1beta, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis. RESULTS: In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater (P < .005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased (P < .05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1beta and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted. CONCLUSION: A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection.

Adult↗
PubMed1998

The extracellular matrix of the human fetal membranes: structure and function.

The human fetal membranes are genetically identical to the fetus and form a highly specialized interface between mother and fetus, of considerable significance to the successful maintenance and termination of pregnancy in the higher vertebrates. Additionally, the upright posture of women presents these tissues with a greater mechanical challenge than in other species. The major extracellular matrix components providing tensile strength and elastic recoil are reviewed, as well as the key enzyme, activator/inhibitor system responsible for their remodelling and breakdown. However, this fails to convey the important concept that the matrix components are bound to each other and to the cells involved in their formation and organization. These matrix components are collectively responsible for the biomechanical properties of the tissue, but they must also be considered as dynamic elements of a broader signalling system, which include hormonal autocrine/paracrine systems. A unifying hypothesis is presented, which attempts for the first time to bring these two facets of the matrix together, which permits a potential coordination of local events at the maternal-fetal interface leading to parturition. In order to understand fully both the normal biology and the pathobiology of these tissues, such integration of the cellular and extracellular signalling pathways must be achieved.

Biomechanical Phenomena↗
PubMed1997

Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity.

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

Amino Acids↗
PubMed1997

An autocrine/paracrine role of human decidual relaxin. I. Interstitial collagenase (matrix metalloproteinase-1) and tissue plasminogen activator.

Decidual and placental relaxins have been proposed as autocrine/ paracrine hormones in the remodeling of collagen in the amnion and chorion in the last weeks of pregnancy. The matrix metalloproteinase-1 (MMP-1) is a key enzyme in the degradation of the interstitial collagens which predominate in the fetal membranes. Distribution of the MMP-1 gene and of the MMP-1 protein was shown by in situ hybridization and immunolocalization, respectively, in amnion, chorion, and decidua collected from patients before the onset of spontaneous labor. The distribution of MMP-1 in the chorionic cytotrophoblast and decidua coincided with that of the human relaxin receptor, detected by tissue section autoradiography in tissues collected at the same stage of pregnancy. Fetal membrane explants were used to study the effect of exogenous human relaxin H2. These responded by a dose-dependent increase in expression of the MMP-1 gene, in its secreted protein, and in its enzyme activity in the medium. A similar dose-dependent increase in the tissue plasminogen activator (tPA) gene and protein upon exposure of the explants to relaxin H2 suggested a coordinated cascade system, resulting in increases in secreted activities of MMP-1, MMP-3 (stromelysin), and MMP-9 (gelatinase B). There was no effect on the genes or proteins for MMP-2 (gelatinase A) or tissue inhibitor of metalloproteinase-1 (TIMP-1), showing the specificity of the response. This coordinated regulation by relaxin H2 of tPA, MMP-1, MMP-3, and MMP-9 would result in more complete degradation of the fetal membrane extracellular matrix components.

Amnion↗
PubMed1997

An autocrine/paracrine role of human decidual relaxin. II. Stromelysin-1 (MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1).

Interstitial collagen types I and III are the predominant collagens in the amniotic and chorionic connective tissues. However, this matrix also contains proteoglycans, fibronectin, laminin, and elastin, which together with the collagens may undergo partial degradation prior to fetal membrane rupture at term. In this study, stromelysin (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were immunolocalized in fetal membranes obtained at term prior to labor. MMP-3 stained the cells of the amniotic epithelium, fibroblasts and macrophages of the amniotic and chorionic matrix, and those of the chorionic cytotrophoblast; there was no staining in the maternal decidua. TIMP-1 showed a similar staining pattern, except that the staining was darker in some amniotic epithelial cells and was present in the maternal decidua. The maternal decidua produces the two human relaxins H1 and H2; the latter, when incubated with explants of human fetal membranes, caused a dose-dependent and significant increase in expression of the MMP-3 gene and its secreted protein into the media. A significant effect of relaxin H2 on 92-kDa gelatinase (MMP-9) gene expression was also shown--an effect requiring poly(A)+ RNA rather than total RNA. Both relaxin H1 and H2 caused a significant increase in secretion of MMP-9 protein and its enzyme activity in the media. The magnitude of the effects of the two relaxins was similar, in contrast to findings from other biological studies in which relaxin H2 was shown to be more active. Neither of the relaxins had any effect on 72-kDa gelatinase (MMP-2) activity or on the TIMP-1 protein or its activity. This study suggests that local relaxins may be involved in the degradation of the complex fetal membrane extracellular matrix and may cause activation of an enzyme cascade resulting in fully activated MMP-9. Such effects could be important in the degradative pathways occurring in the amnion and chorion in the peripartal period.

Blotting, Northern↗
PubMed1997

Developmental regulation of the human relaxin genes in the decidua and placenta: overexpression in the preterm premature rupture of the fetal membranes.

The decidua and placenta synthesize the human relaxins, termed H1 and H2, believed to be involved in collagen remodeling in the amnion and chorion in an autocrine/paracrine manner. The developmental regulation of the relaxin genes was quantitated in normal pregnancy by in situ hybridization histochemistry with six 48-mer oligonucleotide probes that detect both relaxin genes. A significant increase in relaxin expression occurred in both decidua (p < 0.01) and placenta (p < 0.05) at 12.5-14.4 wk gestation, with the mean peak value in the placenta more than double that of the decidua, suggesting a coordinate regulation of the relaxin genes. At term after spontaneous labor and delivery, a marginal increase in both decidual and placental relaxin gene expression occurred. Given these normal data, three abnormal preterm situations were investigated: 1) premature uterine contractions without prior rupture of the membranes, 2) premature rupture of the fetal membranes (PPROM), 3) cesarean section for medical reasons with intact membranes and no uterine contractions. Tissues showing intrauterine infection were eliminated. Significantly more relaxin was expressed in the preterm decidua from patients with PPROM when compared to patients in group 1 (p < 0.02) or group 3 (p < 0.008). These data were confirmed by Northern analysis with a relaxin cRNA probe. The placental tissues after PPROM also had a significantly higher and a uniform overexpression of relaxin in the placental syncytiotrophoblast. Tissues collected at term, in comparison, showed no such increases in decidua or placenta.

Blotting, Northern↗