217 THE EXPRESSION OF β-OXIDATION RELATING GENES UNDER HYPOXIC STRESS IN HUMAN PLACENTAL CHORIOCARCINOMA CELL (BeWo)

2015 ◽  
Vol 27 (1) ◽  
pp. 198
Author(s):  
E.-K. Shin ◽  
E.-B. Jeung
Keyword(s):  
Hypoxic Condition ◽  
Bewo Cell ◽  
Hypoxic Stress ◽  
Experimental Conditions ◽  
Fluorescent Intensity ◽  
Peripheral Tissues ◽  
Bewo Cells ◽  
Normoxic Control ◽  
La Jolla

Preeclampsia (PE) is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. Hypoxia can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy in conjunction with the breakdown of glucose, fats, and some amino acids. The aim of the study was to investigate the question of whether hypoxic stress is involved in β-oxidation of human placental BeWo cells. One of the β-oxidation related genes, ACADVL was detected by gene-fishing technology using the preeclamptic placenta of human. We conducted in vitro experiments to confirm a preliminary study by inducing hypoxic stress in the human placental BeWo cell. BeWo cells were cultured at 37°C in a 20% O2, 5% CO2 humidified tissue culture incubator. And then we induced hypoxic stress in BeWo cell cultured under 1% O2, 5% CO2, and balanced with N2. The BeWo cells were divided into three groups: normoxia, hypoxia 24 h, and hypoxia 48 h. The expression of β-oxidation related genes (ACADVL, EHHADH, HADH, ACAA) were observed under hypoxic condition in BeWo cells by using real-time RT-PCR. Relative quantification with HPRT1 was based on the comparison of CT at a constant fluorescent intensity. The amount of transcript is inversely related to the observed CT, and for every 2-fold dilution in the transcript, CT is expected to increase by 1. Relative expression was calculated using the equation R = 2–(CTsample – CTcontrol). Data were analysed with a nonparametric one-way ANOVA, followed by Tukey's test for multiple comparisons. All statistical analyses were performed using GraphPad Prism software (v 4.0, GraphPad Software, La Jolla, CA, USA). P-values <0.05 were considered statistically significant. The expression of a gene known as a biomarker for hypoxia, HIF-1a, was significantly increased in BeWo cells which induced preeclampsia. The elevated level of HIF-1a indicates that our experimental conditions closely mimicked preeclampsia. The β-oxidation related genes, ACADVL, EHHADH, and HADH expressions were significantly increased under hypoxic condition in BeWo compared with normoxic control. These results indicate that changes of β-oxidation related genes observed under hypoxic BeWo cells are similar to ones associated with preeclampsia, and the expression of β-oxidation related genes were up-regulated by hypoxic stress. They might be involved in pathogenesis of preeclampsia during gestation. Taken together, increase of β-oxidation-related genes under hypoxic stress may cause a compensation of energy metabolism deficiency through lipid metabolism.

113 EXPRESSION OF BETA-OXIDATION-RELATED GENES IN PREECLAMPSIA-LIKE MODEL UNDER HYPOXIC CONDITION IN VIVO AND IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 187
Author(s):  
M. H. Lee ◽  
E.-K. Shin ◽  
H. Y. Kang ◽  
J.-U. Hwang ◽  
E.-B. Jeung
Keyword(s):  
Hypoxic Condition ◽  
Hypoxic Stress ◽  
Beta Oxidation ◽  
Experimental Conditions ◽  
Peripheral Tissues ◽  
Bewo Cells ◽  
In Vivo Experiments ◽  
Mouse Placenta

Preeclampsia (PE) is a disorder of pregnancy characterized by high blood pressure and large amounts of protein in the urine. Preeclampsia is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. The hypoxic condition during the pregnancy can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy. As an expression of beta-oxidation-related genes, ACADVL was detected by gene-fishing technology using the placenta of human. We conducted in vitro and in vivo experiments to confirm preliminary study by inducing hypoxic stress in the BeWo cells and mice placenta. BeWo cells were cultured at 37°C under 1% O2, 5% CO2, and balanced with N2. Pregnant mice were maintained from GD 6.5 to 17.5 under 11% O2, 5% CO2, and balanced with N2. The expression of beta-oxidation related genes (ACADVL, EHHADH, HADH, ACAA1) were observed under hypoxic condition at mRNA and protein levels. The expression of genes known as biomarkers for hypoxia, HIF-1α, was increased in BeWo cells and mouse placenta, which induced PE. The beta-oxidation-related genes ACADVL expression was significantly increased by hypoxic stress both BeWo cells and mouse placenta. The elevated level of HIF-1α indicates that our experimental conditions closely mimicked PE. These results indicate that changes of beta-oxidation-related genes are correlated with PE induced hypoxic condition.

Ultrastructure of Choriocarcinoma in Vitro

1969 ◽  
Vol 27 ◽  
pp. 362-363
Author(s):  
John C. Garancis ◽  
R. A. Pattillo
Keyword(s):  
Cell Line ◽  
Physiological Function ◽  
Cell System ◽  
Bewo Cell ◽  
Bewo Cells ◽  
Gestational Choriocarcinoma ◽  
Bewo Cell Line

Growth of cell system (BeWo-cell line) derived from human gestational choriocarcinoma has been established and continuously maintained in-vitro. Furthermore, it is evident from the previous studies that this cell line has retained the physiological function of the placental trophoblasts, namely the synthesis of human chorionic gonadotrophil(HCG).The BeWo cells were relatively small and possessed single nuclei, thus indicating that this cell line consists exclusively of cytotrophoblasts. In some instances cells appeared widely separated and their lateral surfaces were provided with numerous microvilli (Fig.1).

313. EPIGENETIC REGULATION OF THE CRH GENE PROMOTER INVOLVES SPECIFIC CpG DE-METHYLATION

2010 ◽  
Vol 22 (9) ◽  
pp. 113
Author(s):  
X. Pan ◽  
C. Abou-Seif ◽  
M. Allars ◽  
Y. Chen ◽  
R. C. Nicholson
Keyword(s):  
Epigenetic Regulation ◽  
Genomic Dna ◽  
Methylation Status ◽  
Gene Promoter ◽  
Bewo Cell ◽  
Gestational Length ◽  
Partial Methylation ◽  
Peripheral Tissues ◽  
Cpg Sites ◽  
Bewo Cells

Corticotropin Releasing Hormone (CRH), is expressed in many regions of the central nervous system and in some peripheral tissues, and plays an important role in determining gestational length. In placenta, a cAMP regulatory site (CRE) is crucial for CRH gene regulation. The promoter of CRH gene has 9 CpG sites, which should be the targets of epigenetic regulation by DNA methylation. The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for the placenta. BeWo cells only produce CRH after exposure to cAMP. The DNA methyl transferase (DNMT) inhibitor 5-aza-cytidine stimulates CRH expression 5-fold in camp treated BeWo cells, indicating the CRH promoter as a target of DNMTs. To evaluate methylation differences of the 9 CpG sites in CRH gene promoter in BeWo cells after treatment with cAMP. Genomic DNA was extracted from BeWo cells treated or not with cAMP. Sodium bisulfite conversion was used to modify the genomic DNA. PCR was used to amplify the CRH promoter region with primers that did not contain CpG sites. The PCR products were cloned and sequenced. The CpG methylation status of each sample was obtained by comparing the sequencing results with the original sequence. In non-stimulated cells (control) CpG -4 was methylated in 50% of the clones and CpG -6 was methylated in 75% of the clones, but the other 7 sites were methylated in every clone. In the cAMP treated cells however there was 100% methylation at CpG sites 6 through 9, but only partial methylation at CpG-1 and 3 (60%), CpG-4 and 5 (40%). Most interestingly, there was no methylation found at CpG-2 in any of the clones from cAMP treated cells, indicating that specific CpG de-methylation around the CRE is required for CRH gene expression.

scholarly journals Fusarium Mycotoxins Disrupt the Barrier and Induce IL-6 Release in a Human Placental Epithelium Cell Line

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 665 ◽  
Author(s):  
Negisa Seyed Toutounchi ◽  
Astrid Hogenkamp ◽  
Soheil Varasteh ◽  
Belinda van’t Land ◽  
Johan Garssen ◽  
...  
Keyword(s):  
Bewo Cell ◽  
Placental Barrier ◽  
Fusarium Mycotoxins ◽  
Barrier Integrity ◽  
Bewo Cells ◽  
Junctional Proteins ◽  
Proinflammatory Responses ◽  
Vitro Model ◽  
Cytotoxicity Effects

Deoxynivalenol, T-2 toxin, and zearalenone, major Fusarium mycotoxins, contaminate human food on a global level. Exposure to these mycotoxins during pregnancy can lead to abnormalities in neonatal development. Therefore, the aim of this study was to investigate the effects of Fusarium mycotoxins on human placental epithelial cells. As an in vitro model of placental barrier, BeWo cells were exposed to different concentrations of deoxynivalenol, zearalenone or T-2 toxin. Cytotoxicity, effects on barrier integrity, paracellular permeability along with mRNA and protein expression and localization of junctional proteins after exposure were evaluated. Induction of proinflammatory responses was determined by measuring cytokine production. Increasing mycotoxin concentrations affect BeWo cell viability, and T-2 toxin was more toxic compared to other mycotoxins. Deoxynivalenol and T-2 toxin caused significant barrier disruption, altered protein and mRNA expression of junctional proteins, and induced irregular cellular distribution. Although the effects of zearalenone on barrier integrity were less prominent, all tested mycotoxins were able to induce inflammation as measured by IL-6 release. Overall, Fusarium mycotoxins disrupt the barrier of BeWo cells by altering the expression and structure of junctional proteins and trigger proinflammatory responses. These changes in placental barrier may disturb the maternal–fetal interaction and adversely affect fetal development.

scholarly journals Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage.

1985 ◽  
Vol 5 (12) ◽  
pp. 3458-3466 ◽  
Author(s):  
J Woolford ◽  
V Rothwell ◽  
L Rohrschneider
Keyword(s):  
Rabbit Antiserum ◽  
Growth Factor Receptor ◽  
Protein Product ◽  
Human Cells ◽  
Cell Protein ◽  
Bewo Cell ◽  
Metabolic Labeling ◽  
Bewo Cells ◽  
Human Choriocarcinoma Cells

The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto-oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140-kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto-oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway.

scholarly journals ZBED1 Regulates Genes Important for Multiple Biological Processes of the Placenta

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 133
Author(s):  
Simone Johansen ◽  
Sofie Traynor ◽  
Malene Laage Ebstrup ◽  
Mikkel Green Terp ◽  
Christina Bøg Pedersen ◽  
...  
Keyword(s):  
Cell Model ◽  
Trophoblast Cell ◽  
Innate Immune ◽  
Bewo Cell ◽  
Biological Processes ◽  
Bewo Cells ◽  
Genes Encoding ◽  
And Function

The transcription factor ZBED1 is highly expressed in trophoblast cells, but its functions in the processes of trophoblast and placental biology remain elusive. Here, we characterized the role of ZBED1 in trophoblast cell differentiation using an in vitro BeWo cell model. We demonstrate that ZBED1 is enhanced in its expression early after forskolin-induced differentiation of BeWo cells and regulates many of the genes that are differentially expressed as an effect of forskolin treatment. Specifically, genes encoding markers for the differentiation of cytotrophoblast into syncytiotrophoblast and factors essential for trophoblast cell fusion and invasion were negatively regulated by ZBED1, indicating that ZBED1 might be important for maintaining a steady pool of cytotrophoblast cells. In addition, ZBED1 affected genes involved in the regulation of trophoblast cell survival and apoptosis, in agreement with the observed increase in apoptosis upon knockdown of ZBED1 in forskolin-treated BeWo cells. In addition, genes implicated in the differentiation, recruitment, and function of innate immune cells by the placenta were affected by ZBED1, further suggesting a role for this protein in the regulation of maternal immune tolerance. In conclusion, our study implicates ZBED1 in major biological processes of placental biology.

Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage

1985 ◽  
Vol 5 (12) ◽  
pp. 3458-3466
Author(s):  
J Woolford ◽  
V Rothwell ◽  
L Rohrschneider
Keyword(s):  
Rabbit Antiserum ◽  
Growth Factor Receptor ◽  
Protein Product ◽  
Human Cells ◽  
Cell Protein ◽  
Bewo Cell ◽  
Metabolic Labeling ◽  
Bewo Cells ◽  
Human Choriocarcinoma Cells

The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto-oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140-kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto-oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway.

scholarly journals The use of BeWo cells as an in vitro model for placental iron transport

2008 ◽  
Vol 295 (5) ◽  
pp. C1445-C1453 ◽  
Author(s):  
Sarah J. Heaton ◽  
John J. Eady ◽  
Mary L. Parker ◽  
Kathryn L. Gotts ◽  
Jack R. Dainty ◽  
...  
Keyword(s):  
Total Dose ◽  
Iron Transport ◽  
In Vitro Model ◽  
Cell Layer ◽  
Bewo Cell ◽  
Apical Surface ◽  
Bewo Cells ◽  
Cell Layers ◽  
Vitro Model

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 ± 0.5% of the total dose was transported after 8 h, equivalent to 38.8 ± 2.1 pmol·cm−2·h−1. Transfer of Tf across the cell layer was much more limited; 2.4 ± 0.2% of the total dose was transported after 8 h, equivalent to 5.0 ± 0.4 pmol·cm−2·h−1. Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

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