A novel trophoblast cell surface antigen has been defined by a monoclonal antibody 5T4, raised following immunisation with wheat germ agglutinin (WGA) purified glycoproteins from deoxycholate (DOC) solubilised human syncytiotrophoblast plasma membrane (StMPM). The distribution of the antigen was determined by indirect immunoperoxidase staining of sections of normal organ and placental tissues as well as immunofluorescence and radiobinding assays with a wide variety of cell lines representing differing normal and tumour cell types. In frozen sections of normal full term placenta, 5T4 is strongly expressed only by the syncytiotrophoblast, some extravillous cytotrophoblast and the amniotic epithelium. The 5T4 antigen is apparently not expressed by any maternal component of the placenta nor is it detected in adult liver, lung, bronchus, heart, testis, ovary, brain, or muscle. The antigen is apparently expressed by several specialised epithelia. Immunoprecipitation of radiolabelled StMPM indicated that 5T4 molecules are glycoproteins of mol. wt of approximately 72 kD on SDS-PAGE. 5T4 antigen is selectively expressed by diverse tumour cell lines, including those of developmental origin. The molecular characteristics, relatively restricted normal tissue distribution and expression by certain tumour cell types make this antigen worthy of future study for use as a diagnostic marker of malignancy.

A 72 kD trophoblast glycoprotein defined by a monoclonal antibody.

Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with sv40 large t antigen

SPECIFIC AIMSThis study was designed to determine whether there is a correlation between the rate of phospholipid synthesis and intracellular accumulation of glucosylceramide (GlcCer), the glycosphingolipid that accumulates in the inherited metabolic disorder Gaucher disease due to defective activity of the lysosomal enzyme glucocerebrosidase. We have already shown that on stimulation of its synthesis or on inhibition of its degradation, GlcCer stimulates neuronal growth and development. Since phospholipids are the major structural components of neuronal membranes, we examined whether GlcCer directly influences the rate of phospholipid synthesis.PRINCIPAL FINDINGS1. GlcCer accumulation stimulates axonal growth and phosphatidylcholine synthesisHippocampal neurons cultured from embryonic rats were incubated for > 4 days with conduritol-B-epoxide (CBE), an inhibitor of lysosomal glucocerebrosidase, or were cultured from a mouse model of Gaucher disease (the Gba−/− mouse). In both cases, an ∼ four- to eightfo...

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fj.02-0149fje

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

The neutral red and methylene blue in vitro cytotoxicity assays were compared under a variety of conditions using normal human ovarian epithelial cells to determine whether either assay is superior for studying cell growth. The results were standardized against a DNA spectrofluorometric assay. Although the assays were equivalent in reflecting cell number, each has specific advantages: while neutral red discriminates between viable and dead cells, the methylene blue assay is more sensitive and easier to perform.

Comparison of the neutral red and methylene blue assays to study cell growth in culture.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa protein that stimulates the growth of endothelial cells [Miyazono, K., et al. (1987) J. Biol. Chem. 262, 4098-4103]. Here, we describe a method to purify large quantities of PD-ECGF from human platelet lysate at a high yield (14% overall recovery). The purification method involves five steps, using high-performance liquid chromatography grade hydroxylapatite and hydrophobic chromatographies as the two final steps. The purified material contained two major components of apparent molecular weight values of 46,000 and 44,000. These components coeluted in a high-resolving reversed-phase chromatography and were found to give similar peptide maps after treatments with staphylococcal V8 protease, suggesting that the 44-kDa form is related to the 46-kDa molecule. Partial tryptic digestion of native PD-ECGF revealed that the molecule contains a trypsin-resistant domain of 37-39 kDa. A rabbit antiserum was produced against the purified material and was found to specifically recognize PD-ECGF in immunoblotting. When added to the cell culture medium, an immunoglobulin fraction of the antiserum neutralized the activity of purified PD-ECGF. Furthermore, it completely neutralized the endothelial cell mitogenic activity of platelet lysate, indicating that PD-ECGF is the only mitogen in platelet lysate for this cell type.

High-yield purification of platelet-derived endothelial cell growth factor: structural characterization and establishment of a specific antiserum.

Factors affecting the reproducibility of a spectrofluorimetric assay for the enumeration of human venous endothelium in culture.

Proliferative response of human venous endothelial cells to medium conditioned by human tumour cells.

A factor that stimulates the proliferation of human umbilical vein endothelial cells has been shown to be present in serum-free medium conditioned by the prior growth of a cell line (1PT) derived from a poorly differentiated bronchial carcinoma. Preliminary characterization of this factor has revealed that it is a heat-labile, acid-stable proteinaceous material, the activity of which is not diminished by treatment with a reducing agent. In its partially purified state it has been shown to be anionic and to be associated with material exhibiting a broad molecular weight range of 35 X 10(3) to 100 X 10(3). It does not bind strongly to heparin-Sepharose and its mitogenic effect on endothelial cells is not potentiated by heparin. These properties suggest that this factor may differ from other previously described tumour-derived endothelial mitogens.

M. Daniel

Papers

Factors affecting the reproducibility of a spectrofluorimetric assay for the enumeration of human venous endothelium in culture.

Proliferative response of human venous endothelial cells to medium conditioned by human tumour cells.

An endothelial cell growth factor derived from human lung carcinoma cells grown in serum-free medium.

Induction of changes in the behaviour of endothelial cells by factors produced by a human tumour cell line