Biomedical subjects

G Neufeld

Publications and source records attributed to G Neufeld.

At least 55 records · Page 3Linked to original sources

PubMed1992

Growth factors, receptor kinases, and protein tyrosine phosphatases in normal and malignant melanocytes.

Normal human melanocyte proliferation and differentiation is dependent on stimulation of one of three growth factor/receptor systems. They are fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and mast cell growth factor (MGF), which activate the FGF receptor, c-Met, and c-Kit, respectively, known to be receptor tyrosine kinases. In contrast, human melanoma cells from primary nodular and metastatic lesions grow autonomously partially because of inappropriate production of basic FGF (bFGF) and continuous activation of the bFGF-receptor kinase. Activation of transmembrane receptor tyrosine kinases in melanocytes stimulates not only proliferation but also the expression of pigmentation. Melanoma cells constitutively express several tyrosyl-phosphorylated proteins that in normal melanocytes are stimulated in response to growth factors. This high level of phosphorylation was not due to either the presence of constitutively active Kit kinase and Met kinase nor to the absence of any of several known protein tyrosine phosphatases. Because bFGF by itself does not transform melanocytes to melanomas, there must be additional cooperating factors that confer the malignant phenotype to pigment cells.

Animals↗

PubMed1992

BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors.

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.

Animals↗

PubMed1992

High levels of biologically active vascular endothelial growth factor (VEGF) are produced by the baculovirus expression system.

Vascular endothelial growth factor (VEGF) is a recently discovered mitogen for endothelial cells in vitro, and a potent angiogenesis promoting factor in vivo. VEGF is secreted from producing cells as a homodimer, binds to specific receptors on the cell surface of endothelial cells, and is produced in four forms as a result of alternative splicing. We have expressed the cDNA encoding the 165 amino-acid long isoform of VEGF in insect cells using the baculovirus based expression vector. We show that infected insect cells secrete large amounts of VEGF. Antibodies directed against a synthetic peptide prepared from human VEGF identify the secreted factor. The baculovirus derived VEGF expressed in insect cells (inVEGF) binds directly to the VEGF receptors inVEGF competes with pure mammalian cells derived [125I]-VEGF for binding to the VEGF receptors that are present on the cell surface of endothelial cells. Furthermore, inVEGF is biologically active and induces the proliferation of human umbilical vein derived endothelial cells.

Animals↗

PubMed1991

PCR-based identification of new receptors: molecular cloning of a receptor for fibroblast growth factors.

Transmembrane tyrosine kinases are involved in the control of cell growth and differentiation by extracellular signals. To enable identification of new receptor tyrosine kinases we developed a method that selectively amplifies segments of receptor genes. The method is based on a combination of polymerase chain reaction (PCR) and hybridization screening and it employs three oligonucleotide primers derived from conserved domains of receptor tyrosine kinases. It yields amplification of receptors' genes and appears to ignore cytoplasmic tyrosine kinases. When applied to RNA from 12.5 days post coitum mouse placenta, this methodology resulted in the detection of several putative or established receptors. Molecular cloning of one of these genes, which is identical to the partially characterized bek gene, identified a transmembrane tyrosine kinase with three immunoglobulin-like domains in the extracellular portion, and a cytoplasmic tyrosine kinase sequence. The isolated cDNA shows remarkable homology to the murine flg gene that encodes a receptor for fibroblast growth factors. Indeed, an antibody directed to the carboxy terminus of the deduced bek protein specifically recognized a receptor for acidic and basic fibroblast growth factors in murine hepatoma cells. We therefore expect that the methodology we developed will enable the study of new receptors in hardly accessible biological systems such as early mammalian embryos or stem cells.

Amino Acid Sequence↗

PubMed1990

Characterization of the receptors for vascular endothelial growth factor.

Vascular endothelial growth factor (vEGF) is a recently discovered mitogen for endothelial cells. It is also a potent angiogenic factor. We have characterized the vEGF receptors of endothelial cells using both binding and cross-linking techniques. Scatchard analysis of equilibrium binding experiments revealed two types of high-affinity binding sites on the cell surfaces of bovine endothelial cells. One of the sites has a dissociation constant of 10(-12) M and is present at a density of 3 x 10(3) receptors/cell. The other has a dissociation constant of 10(-11) M, with 4 x 10(4) receptors/cell. A high molecular weight complex containing 125I-vEGF is formed when 125I-vEGF is cross-linked to bovine endothelial cells. This complex has an apparent molecular mass of 225 kDa. Two other faintly labeled complexes with apparent molecular masses of 170 and 195 kDa also are detected. Reduction in the presence of dithiothreitol causes a substantial increase in the labeling intensity of the 170- and 195-kDa complexes, suggesting that these complexes are derived from the 225-kDa complex by reduction of disulfide bonds. The labeling of the vEGF receptors was inhibited by an excess of unlabeled vEGF but not by high concentrations of several other growth factors. Suramin and protamine, as well as several species of lectins, inhibited the binding. The expression of functional vEGF receptors was inhibited when the cells were preincubated with tunicamycin, indicating that glycosylation of the receptor is important for the expression of functional vEGF receptors. Pretreatment with swainsonine on the other hand, did not prevent formation of functional receptors. However, the mass of the 225-kDa complex is decreased by 20 kDa when 125I-vEGF is cross-linked to swainsonine-treated endothelial cells.

Animals↗

PubMed1990

Basic fibroblast growth factor accumulates in the nuclei of various bFGF-producing cell types.

The intracellular localization of basic fibroblast growth factor (bFGF) was studied in BHK-21 cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei of untransfected cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti-bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin-sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK-21 cells. The mitogenic activity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear. These findings suggest that some biological activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF to DNA.

Adrenal Glands↗

PubMed1990

Expression of basic fibroblast growth factor in the nervous system of early avian embryos.

Basic fibroblast growth factor (bFGF) promotes the survival of a subpopulation of non-neuronal cells developing from trunk neural crest. It was therefore important to determine whether this factor is present in the nervous system at early developmental stages. Immunocytochemistry using specific polyclonal and monoclonal antibodies was combined with three highly sensitive assays: bFGF-induced proliferation of bovine adrenal cortex-derived capillary endothelial cells (ACE), a radioimmunoassay for bFGF (RIA) and Western blot analysis. bFGF immunoreactivity was localized to the cytoplasm of neuroepithelial cells derived from embryonic day 2 (E2) quail neural tubes and cultured for one day in a chemically defined medium. Specific staining was observed in young sensory neurons in cultures of neural crest clusters as well as in a subpopulation of non-neuronal cells. In cultured E7 dorsal root ganglia, immunostaining was confined to neuronal cell bodies and fibers. In situ, staining of spinal cord and ganglionic neurons appeared on E6 and increased in intensity towards E10. Various mesoderm-derived structures such as the limb buds, the mesenchyme dorsal to the neural tube, the vertebral muscles and cartilage showed specific staining patterns in addition to neural tissue. In agreement with the results of immunocytochemical studies, 1.4ng bFGF per mg protein was detected in spinal cord extracts by RIA as early as E3, its concentration increased to 8.0 ng mg-1 on E5 and then to a maximum of 18.0 ng mg-1 protein on E10, this was followed by a subsequent decrease in concentration in older embryos. On the other hand, high levels of bFGF were present in vertebral tissues from E10 onwards. Extracts of immunopositive tissues were subjected to heparin-Sepharose affinity chromatography and eluted in a stepwise salt gradient. Fractions that eluted from the columns at 2 M NaCl contained a bFGF-like protein as revealed by their ability to stimulate the proliferation of ACE cells and by Western blot analysis. These data demonstrate that bFGF is expressed during early nervous system development in both central and peripheral neurons.

Animals↗

PubMed1990

The murine flg gene encodes a receptor for fibroblast growth factor.

The human flg gene has been previously shown to encode a tyrosine kinase whose transcription in endothelial cells is regulated by the fibroblast growth factor (FGF). We report the cloning and sequencing of the murine flg which revealed that the full length transcript encodes a transmembrane receptor-like protein. The extracellular portion is composed of three homologous immunoglobulin-like domains, resembling the organization of the receptor for interleukin-1, and the cytoplasmic domain displays unique organization of tyrosine kinase sequences. An anti-peptide antibody directed to the carboxy-terminus of the flg protein recognized three fibroblast-cell proteins (p150, p130 and p105), that undergo phosphorylation in the immune-complex. Moreover, the antibody specifically immunoprecipitated two proteins (p145 and p120) that were covalently crosslinked to basic FGF, but only one (p155) of the two molecular forms of the receptor for acidic FGF. In situ hybridization analysis of mouse embryos (12.5 days p.c.) indicated that flg is transcribed in the prospective brain, areas of the face and the vertebral column. The latter exhibited progressive cranio-caudal concentration into the intervertebral discs. Taken together, these results strongly suggest that the flg gene encodes a receptor for fibroblast growth factor(s).

Amino Acid Sequence↗

PubMed1989

Cardiogenic mixing: mechanisms and experimental evidence in dogs.

Cardiogenic mixing was studied in seven anaesthetized closed-chest dogs undergoing mechanical ventilation by comparing single-breath washout of two poorly soluble inert gases of widely differing diffusivities (helium (He) and sulphur hexafluoride (SF6)) in normal conditions with the heart beating (control), and during reversible temporary myocardial arrest (heart arrest). Cardiac arrest of approximately 20 s duration was induced repeatedly (8-15 times) by intracoronary injection of acetylcholine (approximately 35 mg) facilitated by a non-occluding 7-French gauge angiographic catheter maintained in the left coronary artery. After equilibration of lung gas with 1% helium and 1% SF6, single breath, constant flow expirograms were recorded in the tracheal tube by mass spectrometry after inspiration of test gas-free air. Series deadspace (VD) and relative alveolar slope (S) (increment of expired partial pressure, normalized to mixed expired-inspired partial pressure difference per increment of expired volume (S = (delta P/(PE-P1]/delta VE litre-1], were determined as indices for intrapulmonary gas mixing. The effects attributable to the action of the heart were quantified by the heart arrest: control ratio of VD and S, which were not significantly different from unity (P greater than 0.05) (VD: 0.95 (SD 0.05) for helium and 0.94 (0.07) for SF6; S:1.03 (0.10) for helium and 1.05 (0.14) for SF6. The He:SF6 ratios of VD and S (0.90 and 0.64, respectively), indicating diffusion dependent separation of gases, also were unaffected by the mechanical action of the heart. The data indicate that convective mixing by the mechanical action of the heart did not significantly enhance intrapulmonary mixing and transport.

Acetylcholine↗

PubMed1988

Identification of the fibroblast growth factor receptor in human vascular endothelial cells.

The fibroblast growth factor (FGF) receptor of human umbilical vein-derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal growth factor, insulin, or transferrin. The lectin concanavalin-A does not inhibit the binding of 125I-bFGF to HUE cell-surface receptors, whereas it inhibits bFGF binding to BHK-21 cell-surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti-angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor.

Affinity Labels↗

PubMed1988

Basic fibroblast growth factor: expression in cultured cells derived from corneal endothelium and lens epithelium.

We have examined the possible expression of fibroblast growth factor in cultured cells derived from bovine lens epithelium and corneal endothelium. Lens epithelial, but not corneal endothelial, cells were found to express the acidic fibroblast growth factor (aFGF) gene, whereas both cell types express the gene encoding basic fibroblast growth factor (bFGF), a related mitogen. Expression of bFGF was further examined. Both corneal endothelial and lens epithelial contain 3.7 and 7.0 k bFGF gene transcript, which are translated into material closely related, if not identical with bFGF. Essentially all of the bFGF-like material is bioactive, i.e. it can stimulate the proliferation of capillary endothelial or corneal endothelial cells and the stimulation is blocked by anti-bFGF antibodies. Our results indicate that bFGF derived from corneal endothelial and lens epithelial cells may act as a paracrine and autocrine growth factor in both cell types. Thus, bFGF present in both cell types may play an important role in the proliferation of injured or transformed ocular tissues.

Animals↗

PubMed1988

Expression of human basic fibroblast growth factor cDNA in baby hamster kidney-derived cells results in autonomous cell growth.

Growth factor over-production by responsive cells might contribute to their autonomous proliferation as well as their acquisition of a transformed phenotype in culture. Basic fibroblast growth factor (bFGF) has been shown to induce transient changes in cell behavior that resemble those encountered in transformed cells. In addition, several types of human tumor cells have been shown to produce bFGF. To determine directly the role that bFGF might play in the induction of the transformed phenotype, we have introduced a human bFGF cDNA expression vector into baby hamster kidney-derived (BHK-21) fibroblasts. One of the BHK transfectants, termed clone 19, expresses the bFGF mRNA and produces biologically active bFGF that accumulates to a high concentration inside the cells. These properties correlate with the ability of the cells to grow in serum-free medium without the addition of exogenous bFGF. Clone 19 cells also proliferated in soft agar, indicating that constitutive expression of the bFGF gene results in a loss of anchorage-dependent growth.

Animals↗

PubMed1988

Basic fibroblast growth factor: expression in cultured bovine vascular smooth muscle cells.

The possible expression of fibroblast growth factor in cultured bovine vascular smooth muscle (VSM) cells derived from bovine adult aortic arch has been examined. VSM cells were found to express the basic fibroblast growth factor (bFGF) gene but not that of acidic fibroblast growth factor (aFGF), a related mitogen. Expression of bFGF was further examined. VSM cells contain 3.7 and 7.0 kilobase bFGF gene transcripts, which are translated into material closely related, if not identical, with bFGF when analyzed by its chromatographic behavior on heparin Sepharose, by immunoblot and by radioimmuno- and radioreceptor assays. Essentially all of the bFGF-like material is bioactive, i.e., it can stimulate the proliferation of capillary endothelial or VSM cells and the stimulation is blocked by anti-bFGF antibodies. Our results indicate that bFGF derived from VSM cells may act as a paracrine and autocrine growth factor for that cell type. Thus, bFGF could play an important role in vivo, in controlling the proliferation of VSM cells during embryonic growth of blood vessels or during atherosclerosis.

Animals↗

PubMed1987

Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells.

Cultured cells derived from bovine retinal pigment epithelium (RPE) express the basic fibroblast growth factor (bFGF) gene and they contain 3.7 and 7.0 kb bFGF gene transcripts which are translated into immunoreactive bFGF of Mr 17,500 and 18,000, respectively. The RPE cell-derived bFGF is bioactive, i.e., it can stimulate the proliferation of capillary endothelial cells and the stimulation of cell proliferation is blocked by anti-bFGF antibodies. We suggest that RPE cell-derived bFGF may be involved in the repair mechanisms following retinal injury and in the intravitreal pseudoneoplastic proliferation of the injured RPE.

Animals↗

PubMed1987

Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells.

Nerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK-21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.

Animals↗

PubMed1987

Protamine sulfate inhibits mitogenic activities of the extracellular matrix and fibroblast growth factor, but potentiates that of epidermal growth factor.

Protamine sulfate, an inhibitor of angiogenesis in vivo, markedly inhibits the ability of angiogenic factors such as acidic or basic fibroblast growth factor (aFGF, bFGF) to stimulate the proliferation in vitro of either BHK-21 cells or vascular endothelial cells. The inhibition is reversible, and cells remain viable even after prolonged exposure to protamine sulfate. Protamine sulfate inhibits the mitogenic effects of both growth factors by preventing them from binding to their common cell surface receptors. It also inhibits the mitogenic activity of the extracellular matrix produced by bovine corneal endothelial cells. This substrate has been shown in previous studies to replace the requirement for FGF of many cell types. In contrast, protamine sulfate potentiates the mitogenic activity of epidermal growth factor (EGF). This indicates that protamine sulfate also acts at cellular sites which are not associated with FGF receptors.

Animals↗

PubMed1987

Fibroblast growth factor: structural and biological properties.

Basic fibroblast growth factor (FGF) and acidic FGF are two closely related peptides that are multifunctional. They control proliferation, differentiation, and various other cellular functions in cells derived from the mesoderm and the neuroectoderm. The structural properties, genomic organization, and biological functions of both peptides in vitro or in vivo are reviewed. Their marked ability to enhance formation of connective tissue and vascular capillaries, as well as their involvement in limb regeneration, suggest several possible therapeutic applications.

Animals↗

PubMed1987

Pituitary follicular cells produce basic fibroblast growth factor.

Cultured monolayers of bovine pituitary follicular cells, which transport ions, contain high amounts of mitogenic activity for endothelial cells which, on the basis of gene expression analysis, heparin-Sepharose elution profile, bioassay, immunoblotting, radioimmunoassay, and radioreceptor assay, has been identified as basic fibroblast growth factor (bFGF). These data indicate that follicular cells may be a major source of bFGF in the pituitary gland. Considering that bFGF has been proposed to play a role in paracrine regulation of pituitary hormone secretion, the data also suggest that these cells may exert important local regulatory functions.

Animals↗