scholarly journals Detection of receptors for sulfated polysaccharides in human placenta by biotinylated probes.

1988 ◽  
Vol 36 (9) ◽  
pp. 1097-1102 ◽  
Author(s):  
P L Debbage ◽  
W Lange ◽  
T Hellmann ◽  
H J Gabius
Keyword(s):  
Developmental Stage ◽  
Human Placenta ◽  
Binding Sites ◽  
Dermatan Sulfate ◽  
Specific Binding ◽  
Cell Types ◽  
Sulfated Polysaccharides ◽  
Stage Of Development ◽  
Different Cell Types ◽  
Biotinylated Probes

Histochemical detection of binding sites for sulfated polysaccharides believed to be important mediators within recognitive interactions was carried out by application of biotinylated probes such as heparin, native and desulfated fucoidan, dermatan sulfate, and two types of carrageenans. The probes were derivatized by mild cyanogen bromide activation and subsequent aminoalkylation to allow incorporation of biotin, inserted with an epsilon-aminocaproic acid spacer to reduce charge-related and steric impediments. Specific labeling could be detected in different cell types of human placenta, dependent on the developmental stage. Sulfated polysaccharides bound predominantly to leucocytes in full-term placenta, whereas demonstration of specific binding sites in decidua, syncytiotrophoblasts, and cytotrophoblasts was restricted primarily to heparin and, less intensely, fucoidan, although not desulfated fucoidan. Heparin binding in the placenta after 8 weeks of gestation was reduced for epithelia that, at this stage of development, revealed carrageenan binding sites. Fucoidan binding was at this developmental stage measurable only for leucocytes. These results provide definite histochemical evidence for the presence and developmental regulation of expression of receptors for sulfated polysaccharides in different cell types of human placenta.

scholarly journals Heparin binding lectin of human placenta as a tool for histochemical ligand localization and isolation.

1991 ◽  
Vol 39 (9) ◽  
pp. 1249-1256 ◽  
Author(s):  
H J Gabius ◽  
B Kohnke-Godt ◽  
M Leichsenring ◽  
A Bardosi
Keyword(s):  
Human Placenta ◽  
Binding Sites ◽  
Specific Binding ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Binding Activity ◽  
Full Term ◽  
Heparin Binding ◽  
Specific Binding Sites

Biotinylated heparin has been used to detect the presence of specific binding sites in sections of human placenta, which has prompted demonstration of expression of lectin activity for this proteoglycan. Purification of this lectin from full-term placenta facilitates the synthesis of its biotinylated derivative, using biotin-amidocaproyl hydrazide, without affecting its activity. It also enables immunization to obtain antibodies. The labeled lectin is shown to bind specifically to nuclear and cytoplasmic locations in various cell types of human placenta, nuclear expression of lectin binding sites being more pronounced at the full-term stage than after 8 weeks of development. The structurally related histone H2B exhibits obvious differences in its binding pattern. The presence of ligands accessible to the lectin whose binding activity can be inhibited by addition of an excess of heparin correlates in most instances with the level of lectin expression detected immunohistochemically. Biochemical information on the nature of the glycohistochemically inferred lectin-specific ligand(s) is obtained by affinity chromatography on resin-immobilized lectin. It leads to isolation of a proteoglycan with similar electrophoretic mobility in agarose-polyacrylamide gel electrophoresis relative to the independently purified heparan sulfate-containing fibronectin binding proteoglycan from human placenta. Both fractions inhibit binding of heparin to the lectin and contain immunologically detected co-purified lectin, emphasizing their ligand properties. Application of labeled tissue lectins in conjunction with lectin-specific antibodies is proposed to obtain valuable insights into the expression of the receptor as well as the ligand part of protein-carbohydrate recognition.

Specific binding of [3H]oxytocin in the female rat brain

1983 ◽  
Vol 61 (9) ◽  
pp. 989-995 ◽  
Author(s):  
B. M. Ferrier ◽  
S.A. McClorry ◽  
A. W. Cochrane
Keyword(s):  
Plasma Membrane ◽  
Limbic System ◽  
Microsomal Fraction ◽  
Specific Binding ◽  
Cell Types ◽  
Time Dependent ◽  
Female Rat ◽  
Ph Optimum ◽  
Rat Tissue ◽  
Different Cell Types

Because of demonstrated effects of oxytocin on some limbic system mediated behaviours, the specific binding of [3H]oxytocin to a plasma membrane containing fraction of rat limbic tissue has been studied. The binding of the microsomal fraction of estrogenized, female rat tissue was time dependent and saturable, with a Bmax of 2.5 × 10−l3 moles per milligram of protein and an apparent KD of 3.53 × 10−8 M, and appeared to show positive cooperativity. The pH optimum of the binding was 6.0, close to the pH optimum for oxytocin – neurophysin binding; however, other results show the two types of binding to be different. The microsomal fraction did not appreciably degrade oxytocin under the conditions used for [3H]oxytocin binding. The distribution in limbic tissue of oxytocin-degrading activity and of individual enzymes capable of degrading oxytocin has been examined and an interplay of enzymes concentrated in different cell types is proposed.

Comparative studies of insulin binding to receptor from adipocytes, hepatocytes, monocytes and erythrocytes from the pig

1988 ◽  
Vol 118 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Elisabeth Hjøllund ◽  
Bjørn Richelsen ◽  
Oluf Pedersen
Keyword(s):  
Steady State ◽  
Receptor Binding ◽  
Specific Binding ◽  
Insulin Binding ◽  
Cell Types ◽  
Target Cells ◽  
Suitable Model ◽  
The Individual ◽  
Different Cell Types ◽  
Specific Insulin

Abstract. We have described the receptor binding of A 14-labelled [125I]insulin to viable adipocytes, hepatocytes, monocytes and erythrocytes from the pig. For all cell types the binding was of high affinity, specific for insulin, the non-specific binding low and degradation of insulin in the medium was minimal. At 24°C, steady state insulin binding was achieved in all four cell types. At 37°C, steady state insulin binding could be measured to adipocytes and hepatocytes. Specific insulin binding levels and receptor affinity for blood and fat cells from the pig are comparable to that in human cells, whereas differences, especially according to affinity, exist between pig and rat cell insulin receptor binding. It is therefore concluded that the pig is a more suitable model for studies of insulin binding in man than rodents. Finally, no correlations between the individual binding levels to the different cell types were observed. Hence, measurement of insulin binding to the easier available blood cells cannot replace studies of insulin binding to target cells of insulin.

scholarly journals Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

1993 ◽  
Vol 13 (6) ◽  
pp. 3706-3713 ◽  
Author(s):  
T Satoh ◽  
W J Fantl ◽  
J A Escobedo ◽  
L T Williams ◽  
Y Kaziro
Keyword(s):  
Growth Factor ◽  
B Cell ◽  
Cell Line ◽  
Binding Sites ◽  
Cell Types ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Ras Protein ◽  
Ras Activation ◽  
Different Cell Types

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types.

scholarly journals Mannose-specific binding sites for horseradish peroxidase in various cells of the rat.

1983 ◽  
Vol 31 (1) ◽  
pp. 78-84 ◽  
Author(s):  
W Straus
Keyword(s):  
Mast Cells ◽  
Horseradish Peroxidase ◽  
Binding Sites ◽  
Specific Binding ◽  
Cell Types ◽  
Sinusoidal Cells ◽  
Binding Reaction ◽  
Specific Binding Sites ◽  
Liver Sinusoidal Cells ◽  
Endogenous Lectins

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in fixed sections of various tissues by a method reported previously. Liver sinusoidal cells, mast cells of lymph nodes, and alveolar macrophages of the lung and skin fibroblasts were main cell types showing mannose-specific binding of HRP. Macrophages, fibroblasts, and mast cells in the connective tissue of other organs also showed the reaction. However, macrophages of the spleen, and cultured 3T3 cells and L-cells did not give the reaction. The specificities of the binding reaction were studied by determining the approximate concentrations of competing sugars that suppressed the specific binding of HRP. It was found that the endogenous lectins in macrophages, fibroblasts, mast cells, and liver sinusoidal cells showed similar specificities toward various carbohydrates. D-Mannose and L-fucose had the highest affinity toward the lectins (competing ability for the binding of HRP). D-Mannose-6-phosphate, N-acetyl-D-glucosamine, D-glucose, D-ribose, and D-arabinose showed intermediate affinity, whereas D-xylose and D-galactose showed low affinity. Polymerized mannose in mannan and glycoproteins rich in mannose groups (invertase and ribonuclease B) showed much higher affinity to the binding sites than free mannose.

scholarly journals Endothelial cells express the interleukin-1 receptor type I

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1262-1267
Author(s):  
D Boraschi ◽  
A Rambaldi ◽  
A Sica ◽  
P Ghiara ◽  
F Colotta ◽  
...  
Keyword(s):  
Binding Sites ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cross Linking ◽  
Type I ◽  
Single Cell Level ◽  
Vascular Cells ◽  
Cell Level ◽  
Myelomonocytic Cells ◽  
Different Cell Types

Interleukin-1 (IL-1) profoundly affects a number of functions of vascular cells. Two distinct IL-1 receptors (IL-1R) are expressed on different cell types: the 80 Kd IL-1RI on T cells and fibroblasts, and the 68 Kd IL-1RII on B cells and myelomonocytic cells. The presence and functionality of IL-1R on vascular cells has been investigated by using polyomatransformed mouse endothelial cell (EC) lines (sEnd.1 and tEnd.1). These cells expressed specific and saturable binding sites for IL-1 (1,273 sites per cell with kd 9.5 x 10(-11) mol/L for sEnd.1, and 771 sites per cell with kd 8.5 x 10(-11) mol/L for tEnd.1, with radioiodinated IL-1 alpha as ligand). Binding of IL-1 was also evident at single cell level by autoradiography. By cross-linking studies, the molecular weight of the IL-1 binding protein on EC was approximately 80 Kd. This was confirmed by the presence in EC of mRNA for the 80 Kd IL- 1RI. The IL-1RI on EC was apparently functional, since EC responded to IL-1 with IL-6 mRNA expression and IL-6 bioactivity production. These results were extended to human EC and vascular smooth muscle cells, which were also found to express mRNA for IL-1RI.

scholarly journals Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Yingshi Ouyang ◽  
Tarique Bagalkot ◽  
Wendy Fitzgerald ◽  
Elena Sadovsky ◽  
Tianjiao Chu ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Human Placenta ◽  
Cell Types ◽  
Maternal Blood ◽  
Transmission Risk ◽  
Rna Seq ◽  
Angiotensin Converting Enzyme 2 ◽  
Real Time Quantitative Pcr ◽  
Different Cell Types

ABSTRACT The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a massive impact on human lives worldwide. While the airborne SARS-CoV-2 primarily affects the lungs, viremia is not uncommon. As placental trophoblasts are directly bathed in maternal blood, they are vulnerable to SARS-CoV-2. Intriguingly, the human fetus is largely spared from SARS-CoV-2 infection. We tested whether the human placenta expresses the main SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin and showed that ACE2 and TMPRSS2 are expressed in the trophoblast rather than in other placental villous cells. While furin is expressed in the main placental villous cell types, we surveyed, trophoblasts exhibit the highest expression. In line with the expression of these entry factors, we demonstrated that a SARS-CoV-2 pseudovirus could enter primary human trophoblasts. Mechanisms underlying placental defense against SARS-CoV-2 infection likely involve postentry processing, which may be germane for mitigating interventions against SARS-CoV-2. IMPORTANCE Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely. However, despite the heightened risk to pregnant women, thus far the transmission risk of COVID-19 to the feto-placental unit seems extremely low. This has been recently attributed to a negligible expression of SARS-CoV-2 entry factors in the human placenta. We therefore sought to explore the expression of the entry factors ACE2 and TMPRSS2 in the different cell types of human placental villi. Using a combination of transcriptome sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), in situ hybridization, and immunofluorescence, we found that trophoblasts, but not the other main villous cell types, express ACE2 and TMPRSS2, with a broad expression of furin. Correspondingly, we also showed that primary human trophoblasts are permissive to entry of SARS-CoV-2 pseudovirus particles.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types

1993 ◽  
Vol 13 (6) ◽  
pp. 3706-3713
Author(s):  
T Satoh ◽  
W J Fantl ◽  
J A Escobedo ◽  
L T Williams ◽  
Y Kaziro
Keyword(s):  
Growth Factor ◽  
B Cell ◽  
Cell Line ◽  
Binding Sites ◽  
Cell Types ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Ras Protein ◽  
Ras Activation ◽  
Different Cell Types

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types.

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