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Biomedical subjects

D S Charnock-Jones

Publications and source records attributed to D S Charnock-Jones.

At least 55 records · Page 3Linked to original sources
PubMed1996

Expression of prostacyclin and thromboxane synthases in placenta and placental bed after pre-eclamptic pregnancies.

Prostacyclin and thromboxane are potent antagonistic regulators of vascular tone and platelet aggregation. In pre-eclampsia, the ratio of their metabolites is decreased. Little is known about the local regulation of intrauterine prostacyclin and thromboxane production in this condition. Placenta and placental bed biopsies were obtained from uncomplicated and pre-eclamptic pregnancies. Prostacyclin synthase (PCS) and thromboxane synthase (TXS) and their mRNA's were localized by immunohistochemistry using monoclonal antibodies and in situ hybridization. Protein and mRNA levels were quantified by immunoblot and RNase protection assay. PCS-like immunoreactivity was found in endothelial cells and leiomyocytes, whereas fetal and maternal macrophages showed positive staining for TXS. Their mRNA was localized to trophoblast and endothelium, and TXS mRNA could also be detected in macrophages. Quantitative analysis showed no significant difference in intrauterine protein or mRNA expression after pre-eclampsia. The prostacyclin and thromboxane production seems to be compartmentalized within the uteroplacental unit. The expression of their synthesizing enzymes might be regulated post-transcriptionally. Additional regulation of prostaglandin production could be metabolically or on the substrate level and requires further elucidation.

Adult↗
PubMed1996

Maternal plasma levels of vascular endothelial growth factor in normotensive pregnancies and in pregnancies complicated by pre-eclampsia.

We have measured the level of vascular endothelial growth factor (VEGF) in maternal plasma during normotensive pregnancy and in pregnancies complicated by pre-eclampsia. VEGF was measured using a competitive enzyme immunoassay. Plasma VEGF was significantly elevated (P < 0.0001) in the pre-eclamptic group (median value 32.7 ng mL-1, range 10.3-64.0), compared with the normotensive group (median value 11.7 ng mL-1, range 6.3-24.3). VEGF is a potent regulator of endothelial cell function. The increased level found in women with pre-eclampsia indicates that VEGF may be involved in the maternal endothelial cell dysfunction associated with this condition. An increase in VEGF, a potent regulator of microvascular permeability, may also contribute to the extravasation of plasma proteins and the subsequent development of proteinuria, both characteristic features of pre-eclampsia.

Adolescent↗
PubMed1996

Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis.

Active endometriosis is characterized by hypervascularization both within and surrounding the implant; therefore the presence of angiogenic factors in the peritoneal environment would be of great importance. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in both physiological and pathological angiogenesis. We sought to determine if VEGF was present in the peritoneal fluid of women with and without endometriosis, and to establish if differences exist between these groups. VEGF was present in all patients sampled. The fluid from patients with endometriosis contained significantly greater amounts of VEGF than controls. Cyclic variations in VEGF concentration were seen in fluid from patients with endometriosis, the VEGF concentration in proliferative phase being significantly higher than in the secretory phase. The concentration of VEGF in this fluid was also significantly higher than that found in the proliferative and secretory phases of women without endometriosis. No cyclic variations in VEGF were seen in the control group. We suggest that elevated levels of VEGF in the peritoneal fluid of patients with endometriosis may be critical in the pathogenesis of endometriosis.

Adult↗
PubMed1996

Localization of VEGF and expression of its receptors flt and KDR in human placenta throughout pregnancy.

Vascular endothelial growth factor (VEGF) is a potent secreted angiogenic growth factor. Its action is mediated through the tyrosine kinase receptors flt and KDR. We here examine, in detail, the distribution of this ligand and its receptors in human placentae throughout gestation. In the first trimester, in-situ hybridization revealed uneven distribution of flt mRNA around the villous trophoblast indicating spatial regulation. Temporal regulation of flt was observed with no flt mRNA expression detected in villi from mid-gestational placenta, while low levels were found in term villi. Extravillous trophoblast was found to contain both mRNA encoding flt and flt-like immunoreactivity throughout pregnancy. In contrast, KDR mRNA was found only in association with endothelial cells. Within the decidua the anti-flt antibody stained multiple cell types during the first trimester of pregnancy but only the extravillous trophoblast later in gestation. VEGF immunoreactivity tended to co-localize with the staining for flt. These results indicate that VEGF may exert an important role within both the placental villi and the maternal decidua in relation to the growth, differentiation and migration of trophoblast and that this is mediated primarily through the spatial and temporal regulation of the flt receptor rather than the KDR receptor.

Endothelial Growth Factors↗
PubMed1996

VEGF mRNA levels in placentae from pregnancies complicated by pre-eclampsia.

OBJECTIVE: To measure the mRNA levels of vascular endothelial growth factor and its receptor in the placenta following delivery after uncomplicated pregnancy and after pregnancies complicated by pre-eclampsia. SETTING: Rosie Maternity Hospital, Cambridge. MATERIAL: Placental biopsies were obtained following delivery by caesarean section in 23 cases of pregnancy presenting at a range of gestational ages with pre-eclampsia. These were compared with biopsies from 20 appropriately matched women with uncomplicated pregnancies. MAIN OUTCOME MEASURE: mRNA levels of vascular endothelial growth factor (VEGF) and its receptor the fins-like tyrosine kinase (flt), were quantified in total RNA isolated from placental biopsies using the RNAse protection assay. The amount of RNA was compared with that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as a standard. Results were expressed as arbitrary optical density units of VEGF/GAPDH and flt/GAPDH. RESULTS: In both control and pre-eclamptic women regression analysis showed that the level of mRNA encoding VEGF declined significantly with gestational age (P < 0.0001). However, levels of VEGF mRNA were significantly lower in the pre-eclamptic women compared with the control women (P < 0.023). CONCLUSIONS: This study provides evidence of an abnormality of growth factor expression in the placenta during pregnancies complicated by pre-eclampsia. Such placentae exhibit deficient growth and differentiation of terminal villi and reduced fetal capillary branching and reduced levels of VEGF could well account for these morphometric changes. This finding provides a molecular explanation for this abnormal placental development and points to VEGF as a factor in the aetiology of pre-eclampsia and its complications.

Adolescent↗
PubMed1996

Messenger RNA encoding thiol protein disulphide isomerase in amnion, chorion and placenta in human term and preterm labour.

OBJECTIVE: To investigate levels of messenger RNA (mRNA) encoding thiol protein disulphide isomerase, in human amnion, chorion and placenta during pregnancy and in relation to term and preterm labour. DESIGN: Amnion, chorion and placenta from 33 women delivered between 24 and 41 weeks of gestation were used in the study. SETTING: Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge Clinical School, Rosie Maternity Hospital, Cambridge. RESULTS: Women who were delivered spontaneously before 30 weeks of gestation had higher levels of mRNA encoding thiol protein disulphide isomerase in placenta and chorion than those who were delivered spontaneously after this time (placenta (P < 0.01, chorion P < 0.01) and compared with those who were delivered by elective caesarean section before 30 weeks of gestation (placenta (P < 0.01, chorion P < 0.05). In the group in whom spontaneous labour occurred, at all gestations studied, there were increased levels of mRNA encoding thiol protein disulphide isomerase in the placenta (P < 0.001) and chorion (P < 0.001) compared with the amnion. CONCLUSION: Changes in the steady state level of mRNA encoding thiol protein disulphide isomerase may play a role in the onset of preterm labour before 30 weeks of gestation.

Adolescent↗
PubMed1996

Characterization and expression of vascular endothelial growth factor (VEGF) in the ovine corpus luteum.

The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2-4), mid- (day 8) and late (days 14-15) stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF (VEGF120, VEGF164, and VEGF188) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2-4 than on day 8 or days 14-15. Densitometric analysis of gels from the RNase protection assay showed that VEGF120 represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.

Amino Acid Sequence↗
PubMed1996

Hepatocyte growth factor/scatter factor and its receptor c-met: localisation and expression in the human placenta throughout pregnancy.

Hepatocyte growth factor (HGF), also known as scatter factor, acts via the c-met receptor resulting in pronounced effects on certain epithelial cells. We hypothesised that HGF would be important in placental development where the trophoblast represents a specialised barrier of epithelial origin. In this paper we examine the expression and production of HGF and its receptor in the human placenta throughout pregnancy. In addition, RT-PCR was undertaken on human embryos to ascertain whether pre-implantation embryonic or trophoblast cells were under the influence of this growth factor. In samples from the first trimester of pregnancy in situ hybridisation with a c-met antisense probe detected message expression in villous cytotrophoblast and in decidual glands but not in extravillous trophoblast. Some c-met expression was detected in cytotrophoblast from the second trimester placentae; this declined to negligible levels by term. Staining with an anti c-met antibody largely confirmed these findings but found relatively strong staining of cytotrophoblast at term. HGF was confined to the villous core throughout pregnancy when examined by both in situ hybridisation and immunohistochemistry. Trophoblast was consistently negative for HGF. Pre-implantation embryos examined by RT-PCR were negative for both c-met and HGF mRNA. These results indicate that the HGF may exert an important influence on cytotrophoblast throughout the process of placental formation and growth.

Blastocyst↗
PubMed1995

Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma.

BACKGROUND: Two thirds of patients with ovarian carcinoma have advanced disease at diagnosis and have poor prognoses because of the presence of highly invasive carcinoma cells and rapidly accumulating ascitic fluid. Vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells, is produced in elevated amounts by many tumors, including ovarian carcinomas. The known human receptors for VEGF, flt and KDR, are both cell surface tyrosine kinases and are expressed predominantly on endothelial cells. Acting through these receptors, VEGF may stimulate angiogenesis and promote tumor progression. PURPOSE: We aimed to clarify the function of VEGF in tumor development by identifying the cells in ovarian carcinoma tissue that express VEGF and its receptors. METHODS: VEGF, flt, and KDR expression was localized by in situ hybridization and immunohistochemistry in frozen sections of primary tumors from five patients with ovarian carcinoma and from metastases of ovarian carcinoma from three different patients. Reverse transcription followed by polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay were used to analyze VEGF, flt, and KDR expression in six epithelial cell lines derived from ovarian carcinoma ascites from five additional patients. RESULTS: Messenger RNAs (mRNAs) encoding VEGF, flt, and KDR were detected in primary ascitic cells and in three of four ovarian carcinoma cell lines examined by RT-PCR. Two novel complementary DNAs that may encode truncated, soluble forms of flt were cloned from one primary source. VEGF levels of 20-120 pM were found in culture media conditioned by the cell lines. Elevated expression of VEGF mRNA was found in all primary tumors and metastases, especially at the margins of tumor acini. VEGF immunoreactivity was concentrated in clusters of tumor cells and patches of stromal matrix. flt immunoreactivity was confined to tumor blood vessels, but flt mRNA was not detected by in situ hybridization. In contrast, KDR mRNA was detected not only in vascular endothelial cells but also in tumor cells at primary malignant sites. CONCLUSIONS: VEGF is expressed by tumor cells in primary and metastatic ovarian carcinoma and accumulates in the stromal matrix. Its receptors, flt and KDR, are expressed by some tumor cells that coexpress VEGF. This is the first localization of KDR expression in nonendothelial cells. IMPLICATIONS: Coexpression of VEGF and KDR by tumor cells in ovarian carcinoma raises the possibility of autocrine stimulation and of therapeutic strategies targeting this receptor-ligand interaction.

Base Sequence↗
PubMed1995

Localization and quantification of vascular endothelial growth factor messenger ribonucleic acid in human myometrium and leiomyomata.

Vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and VEGF peptide were studied in human myometrium and leiomyomata. Amplification of complementary DNA from myometrium and leiomyomata by the polymerase chain reaction revealed three species of mRNA encoding VEGF: VEGF189, VEGF165, and VEGF121. In situ hybridization demonstrated VEGF mRNA expression throughout the smooth muscle cells of myometrium and leiomyomata. VEGF-like immunoreactivity was also detected in these tissues by immunocytochemistry. Quantification of VEGF mRNA using an RNAse protection assay demonstrated that in normal myometrium, levels of VEGF mRNA are significantly higher in the secretory phase than in the proliferative phase of the cycle. Leiomyomata did not have significantly different levels of VEGF mRNA compared with normal myometrium. In untreated leiomyomata, there was no significant difference between VEGF mRNA levels in the proliferative and secretory phases of the cycle. Leiomyomata from women treated with a GnRH anlog did not have significantly different levels of VEGF mRNA from untreated leiomyomata.

Adult↗
PubMed1995

Localization of vascular endothelial growth factor and its receptor, flt, in human placenta and decidua by immunohistochemistry.

Vascular endothelial growth factor is a secreted angiogenic growth factor the mRNA of which is present in the placenta. The mRNA encoding the vascular endothelial growth factor receptor, flt, has also been demonstrated in placenta, with trophoblast appearing to be a novel site of flt expression. We investigated the expression of both vascular endothelial growth factor and flt-like immunoreactivity in first trimester and term placentae. In the first trimester, vascular endothelial growth factor immunoreactivity was localized to placental macrophages (Hofbauer cells), and in decidua, to glandular epithelium and maternal macrophages. In the term placenta, vascular endothelial growth factor immunoreactivity was present in extravillous trophoblast and in extracellular material. Flt immunoreactivity was demonstrated on extravillous trophoblast in first trimester and term, and on Hofbauer cells within placental villi. This complex pattern of both vascular endothelial growth factor and flt-like immunoreactivity suggests that vascular endothelial growth factor may be involved not only in the regulation of placental angiogenesis, but also in trophoblast invasion.

Decidua↗
PubMed1994

Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction.

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/micrograms total RNA. In medulla these values were 0.46 and 1.16 amol/micrograms, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens.

Base Sequence↗
PubMed1994

Extension of incomplete cDNAs (ESTs) by biotin/streptavidin-mediated walking using the polymerase chain reaction.

In the last 2 years thousands of new partial cDNAs or expressed sequence tags (ESTs) have been identified by single pass sequencing methods. It is expected that this number will further increase in order to help to isolate all human genes. However, the scientific value of partial cDNA fragments is limited unless they are used as tools for isolating and sequencing their full length parent molecules. Conventional library screening methods are tedious and not very effective in achieving this goal. We present a modified PCR technique which allows rapid isolation of the ends of partial cDNA fragments in vitro using a biotin/streptavidin capture procedure. Our method has several advantages over the RACE technique, is very specific, and allows to frequently sequence the final product directly without subcloning. We also show that cDNA walks can be obtained from partial sequences as short as 26 bp.

Bacterial Proteins↗
PubMed1994

Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells.

Vascular endothelial growth factor (VEGF; also known as vascular permeability factor) is a secreted angiogenic growth factor. It is highly specific for endothelial cells, and its receptor, the fms-like tyrosine kinase (flt), has been localized only to endothelial cells in vivo. Here we describe the expression of mRNA encoding flt in human trophoblast as revealed by in situ hybridization. This mRNA is highly expressed in the cytotrophoblast shell and columns and also highly expressed by the extravillous trophoblast (EVT) in the maternal decidua both in the first trimester and at term. The trophoblast-like choriocarcinoma cell line BeWo also expresses this receptor and the related receptor, kinase domain-containing receptor (KDR), which is also a receptor for VEGF. Treatment of the cell line BeWo with VEGF165 stimulated 3H-thymidine incorporation and tyrosine phosphorylation of MAP (mitogen-activated protein) kinase in a time- and dose-dependent fashion. This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.

Base Sequence↗
PubMed1994

Quantification of messenger ribonucleic acid for epidermal growth factor in human myometrium and leiomyomata using reverse transcriptase polymerase chain reaction.

Epidermal growth factor (EGF) mRNA was quantified in samples of human myometrium, untreated leiomyomata, and leiomyomata from patients treated with a GnRH analog. Quantitative reverse transcriptase-polymerase chain reaction, using a synthetic internal standard, was applied to determine levels of EGF mRNA. In myometrium from uteri with no leiomyomata, levels of EGF mRNA did not differ between the proliferative and secretory phase of the cycle. Leiomyomata from women who had received no drug therapy had significantly higher amounts of EGF mRNA than myometrium from a normal uterus, but only in the secretory phase of the cycle. In the proliferative phase, leiomyomata did not have different amounts of EGF mRNA compared to normal myometrium. Untreated leiomyomata in the secretory phase of the cycle, but not those in the proliferative phase, had significantly more EGF mRNA than leiomyomata from women who had received treatment with a GnRH analog. These findings suggest that EGF is important in leiomyomata development, but imply that its production is only increased during the secretory phase of the cycle. This challenges the hypothesis that EGF production in leiomyomata is mediated by estrogen and raises the possibility that progesterone may be the more important hormone in fibroid growth.

Adult↗
PubMed1994

Leukaemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at this time.

Maternal endometrial leukaemia inhibitory factor (LIF) is required for successful implantation in mice. Mice with homozygous deletions in this gene fail to support implantation. The localization of immunoreactive LIF and the concentration of the mRNA encoding human LIF in normal endometrium during the menstrual cycle were investigated. The amount of RNA was low or undetectable in the proliferative phase but increased by approximately six times in the mid- to late secretory phase. The protein can only be detected by immunocytochemistry in glandular epithelium in the mid- or late secretory phase. To investigate the possible target for the endometrial LIF, we undertook reverse transcription-PCR analysis of early human embryos to determine whether they contain the mRNA encoding the LIF receptor. This study indicated that at the time of implantation in humans, the maternal endometrium produces LIF and that the blastocyst expresses LIF receptor mRNA and therefore may be capable of responding to this signal.

Base Sequence↗
PubMed1993

Alternatively spliced mRNAs for human endothelin-2 and their tissue distribution.

The cDNA for Endothelin-2 (ET-2) has been previously cloned and characterised; however, ET-2 remains the least studied of the endothelin isopeptides and little is known of its function and location. In the present study reverse transcriptase-polymerase chain reaction revealed the presence of seven alternatively spliced mRNA variants encoding ET-2, with a specific pattern of distribution in various human tissues. Computer alignment and analysis of the DNA sequences demonstrated alternative splicing of five exons of 52, 169, 123, 99 and 174 base pairs, in the carboxy terminal region of the mRNA encoding preproET-2. This region contains sites for the post-transcriptional processing of preproET-2 into mature ET-2, therefore we postulate that post-transcriptional processing may be disrupted or altered in these variants.

Alternative Splicing↗