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J Sasse

Publications and source records attributed to J Sasse.

At least 55 records · Page 3Linked to original sources
PubMed1991

Subcellular distribution of basic fibroblast growth factor in human hepatoma cells.

The subcellular distribution of endogenous basic fibroblast growth factor (bFGF) was studied in the human hepatoma cell line, SK Hep-1. Basic FGF was demonstrated in cytosol, nuclei, and membranes by purification from each subcellular fraction using ion-exchange chromatography and heparin-affinity chromatography, and by the detection of bFGF-immunoreactive proteins on Western blots of heparin-affinity purified samples. About 65% of bFGF bioactivity was present in cytosol, 17% in nuclei, and 18% in membranes. Antisera raised against either recombinant 18 kDa bFGF or a bFGF N-terminal extension peptide showed that cytosol contained bFGF of mainly Mr 18,000 whereas nuclei and membranes contained three forms of bFGF of Mr 18,000, 22,500, and 24,000. Mitogenic activity in nuclei was chromatin-associated and required 0.6 M NaCl or 100 micrograms/ml heparin for maximal release. Membrane-bound activity was released by 0.6 M NaCl but not by heparin. The finding that endogenous bFGF proteins are present in various subcellular compartments suggests that bFGF may have additional biological roles at these sites.

Amino Acid Sequence↗
PubMed1990

Isolation, characterization, and localization of heparin-binding growth factors in the heart.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are angiogenic polypeptide mitogens for cells of mesodermal and neuroectodermal origin. In this report we describe the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF. Further evidence includes (a) prevention of mitogenicity by protamine and by anti-bFGF, (b) displacement of 125I-bFGF from cell membranes, and (c) stimulation of capillary endothelial cell migration. Specific immunohistochemistry localized bFGF to endothelial cells and, surprisingly, to cardiac myocytes, with almost no immunoreactivity in smooth muscle cells. These peptides may function in cardiac embryogenesis, hypertrophy, atherogenesis, angiogenesis, and wound healing, and may also have endocrine, neurotropic, or vasomotor functions.

Aged↗
PubMed1990

Species-specific high molecular weight forms of basic fibroblast growth factor.

bFGF was extracted from either mouse, rat and human cell lines or mouse, rat bovine and human brain tissue and partially purified by cation exchange chromatography and heparin-affinity chromatography. When the heparin-affinity purified proteins were probed on Western blots with antisera against either a highly conserved internal bFGF sequence or recombinant 18 kDa bFGF, species-specific forms of bFGF were detected. bFGF proteins from rat and mouse sources were of apparent molecular weight 18,000, 21,500 and 22,000 whereas those from human sources were of 18,000, 22,500 and 24,000. Bovine bFGF proteins were similar to the multiple human bFGFs. The 22.5 kDa and 24 kDa proteins from human cells were also recognized by an antibody specific for the N-terminally extended forms of human bFGF, whereas this antibody failed to detect 18 kDa bFGF. We showed that the differences in molecular weight between human and rat bFGFs are consistent with the predicted ATG (methionine) or alternative CTG (leucine) translational start sites in the 5' upstream sequences of bFGF cDNAs. In addition we show that, irrespective of the species of origin, the larger bFGF proteins may be separated from 18 kDa bFGF by Mono S chromatography.

Amino Acid Sequence↗
PubMed1989

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation.

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.

Animals↗
PubMed1988

Increased levels of fibroblast growth factor-like activity in urine from patients with bladder or kidney cancer.

Growth factor activity was partially purified from human renal tumors and a human bladder cancer cell line by heparin-Sepharose chromatography. This activity stimulated bovine capillary endothelial cell proliferation and DNA synthesis in BALB/c 3T3 cells. Partially purified growth factor preparations from these tumors contained a protein with an approximate molecular weight of 17,000 which was recognized by a polyclonal antiserum raised against a peptide fragment of basic fibroblast growth factor (FGF). This growth factor activity appears to be related to basic fibroblast growth factor. Measurement of FGF-like activity in 50 urine samples from 32 adult males showed that 55% (6 of 11) of the urine samples from patients with bladder cancer and 100% (7 of 7) of the urine samples from patients with kidney cancer contained activity equivalent to more than 20 ng of basic FGF/h of urine production. In contrast, only 6% (2 of 32) of the urine samples from controls, patients with a benign disease, or patients with a history of bladder or kidney cancer contained this level of growth factor activity. These results suggest that patients with bladder or kidney cancer release an FGF-like factor into urine which may be used as a marker for these tumors.

Adenocarcinoma↗
PubMed1988

A growth factor in bovine and human testes structurally related to basic fibroblast growth factor.

Homogenates of human testes, epididymides and prostate, and calf testes and epididymides are mitogenic for cultured human foreskin fibroblasts. The growth factors appear similar in that they are inactivated by boiling and acid, but not by treatment with reducing agent. The growth factor in human and bovine testes was partially purified from tissue homogenates, prepared in high ionic strength buffer (pH 7.6) containing protease inhibitors, by ammonium sulfate precipitation and two cycles of heparin-Sepharose chromatography. The growth factor in calf testes was also partially purified from tissue extracted in ammonium sulfate without protease inhibitors, acidified to pH 4.5, and precipitated by ammonium sulfate followed by two cycles of heparin-affinity chromatography. A predominant 17,500 molecular weight (MW) growth factor was identified from alkaline homogenates of human and calf testes by its reactivity with antisera prepared against synthetic peptides whose sequences corresponded to residues 1-12 (amino-terminal), 33-43 (internal) and 136-145 (carboxy-terminal) of bovine basic fibroblast growth factor (bFGF). A slightly smaller 16,600 MW peptide from acidic extracts of calf testes also reacted with antisera to the three synthetic peptides. A 15,500 MW peptide, lacking immunoreactivity with antiserum to the amino-terminal synthetic peptide, was also seen. These findings suggest that a growth factor is present in human and calf testes that is structurally related to bFGF. The structure of the growth factors appears to be altered during the isolation procedure.

Aged↗
PubMed1988

Characterization of growth factors derived from the rat ventral prostate.

Tissue homogenates of rat ventral prostate were examined for growth factor activity using a fibroblast mitogenesis assay. G-75 Sephadex gel filtration separated the growth factor activity into two peaks. A broad first peak contained 98% of the protein and several growth factor moieties. A smaller second peak (MW 6,000) contained epidermal growth factor (EGF) as determined by binding in both a competitive receptor binding assay and a radioimmunoassay using anti-mouse epidermal growth factor (anti-mEGF). The broad first peak also contained substantial amounts of EGF-like activity as higher MW forms of EGF. The broad first peak was further fractionated by heparin-Sepharose affinity chromatography. A major fraction with growth factor activity eluted at 1.5 M NaCl and this fraction was shown to contain bFGF by immunostaining with antisera prepared against synthetic peptides corresponding to amino acid sequences 1-12 (amino terminal), 33-43 (internal), and 136-145 (carboxy terminal) of basic fibroblast growth factor (bFGF). EGF-like and bFGF-like molecules account for the major growth factor activity in the rat ventral prostate.

Animals↗
PubMed1988

Identification of a tumor cell receptor for VGVAPG, an elastin-derived chemotactic peptide.

Extracellular matrix proteins and their proteolytic products have been shown to modulate cell motility. We have found that certain tumor cells display a chemotactic response to degradation products of the matrix protein elastin, and to an elastin-derived peptide, VGVAPG. The hexapeptide VGVAPG is a particularly potent chemotaxin for lung-colonizing Lewis lung carcinoma cells (line M27), with 5 nM VGVAPG eliciting maximal chemotactic response when assayed in 48-microwell chemotaxis chambers. Binding of the elastin-derived peptide to M27 cells was studied using a tyrosinated analog (Y-VGVAPG) to allow iodination. Scatchard analysis of [125I]Y-VGVAPG binding to viable M27 tumor cells at both 37 and 4 degrees C indicates the presence of a single class of high affinity binding sites. The dissociation constant obtained from these studies (2.7 X 10(-9) M) is equivalent to the concentration of VGVAPG required for chemotactic activity. The receptor molecule was identified as an Mr 59,000 species by covalent cross-linking of the radiolabeled ligand to the M27 tumor cell surface and subsequent analysis of the cross-linked material by electrophoresis and size-exclusion high performance liquid chromatography. These results suggest that M27 tumor cell chemotaxis to VGVAPG is initiated by high affinity binding of the peptide to a distinct cell surface receptor.

Animals↗
PubMed1988

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies.

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.

Animals↗
PubMed1988

A heparin-binding angiogenic protein--basic fibroblast growth factor--is stored within basement membrane.

The basement membranes of bovine cornea are found to contain an angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), as determined by heparin-affinity chromatography, immunoblotting, immunofluorescence, and stimulation of capillary endothelial cell proliferation. The growth factor appears to be bound to heparan sulfate and is released from the cornea by treatment with heparin, a hexasaccharide heparin fragment, heparan sulfate, or heparanase, but not by chondroitin sulfate or chondroitinase. These findings indicate that basement membranes of the cornea may serve as physiologic storage depots for an angiogenic molecule. Abnormal release of this growth factor could be responsible for corneal neovascularization in a variety of ocular diseases. Physiologic and pathologic neovascularization in other tissues may also be initiated by release of stored angiogenic factors from the basement membrane. The sequestration of angiogenic endothelial mitogens in the basement membrane may be a general mechanism for regulating their accessibility to vascular endothelium.

Angiogenesis Inducing Agents↗
PubMed1987

Prostatic growth factor: purification and structural relationship to basic fibroblast growth factor.

Prostatic growth factor (PrGF) was purified from alkaline homogenates of human benign prostatic hyperplastic tissue by a combination of ammonium sulfate precipitation, heparin affinity chromatography, and cation-exchange chromatography. The 17,600-dalton, basic (pI 10.2) PrGF is related to basic fibroblast growth factor (bFGF) since antisera raised against synthetic peptides with sequence homologies corresponding to an internal peptide and amino- and carboxyl-terminal peptides of bFGF react with the growth factor. The growth factor appears larger than bFGF, suggesting that additional amino-terminal sequences may be present as a result of alkaline extraction in the presence of protease inhibitors.

Amino Acid Sequence↗
PubMed1987

Amino-terminal sequence of a large form of basic fibroblast growth factor isolated from human benign prostatic hyperplastic tissue.

Homogenization of human benign prostatic hyperplastic tissue in high ionic strength alkaline buffer containing protease inhibitors resulted in the isolation of a 17,400 molecular weight growth factor. When tissue was homogenized in ammonium sulfate at pH 4.5 without protease inhibitors a smaller, 16,600 dalton, growth factor was isolated. Both growth factors reacted with antisera against synthetic peptides whose sequences corresponded to the amino-terminal (1-12), Internal (33-43) and carboxyl-terminal (135-145) portions of basic fibroblast growth factor (bFGF). This suggested that the smaller growth factor was not a truncated form of (1-146) bFGF and that the larger growth factor may contain additional sequences. Amino-terminal sequencing showed the larger growth factor to have the sequence: Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu-Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly- Ala-Phe-Pro-. These results show that the larger growth factor is an 8 amino acid extended from of (1-146) bFGF and it is likely that the smaller growth factor is a proteolytic cleavage product of the larger growth factor produced during the extraction procedure.

Amino Acid Sequence↗
PubMed1987

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted.

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.

Animals↗
PubMed1987

Purified monocyte-derived angiogenic substance (angiotropin) stimulates migration, phenotypic changes, and "tube formation" but not proliferation of capillary endothelial cells in vitro.

Activated monocytes (macrophages, histiocytes) induce the formation of new blood vessels by secretion product(s). From conditioned serum-free media of porcine peripheral monocytes treated with concanavalin A, a substance with very strong angiogenic activity in vivo, designated as angiotropin, has been isolated and purified to homogeneity. We investigated the biological action of the monocyte-derived angiogenic substance on cultured capillary and large vessel (aorta) endothelial cells and on 3T3 fibroblasts, mimicking steps of the angiogenic pathway in vitro. We found that angiotropin does not stimulate the proliferation of capillary endothelial and 3T3 cells; however, in concentrations less than 1 ng/ml, it enhances random migration of capillary endothelial cells but not of 3T3 cells. On confluent monolayers of capillary and aortic endothelial cells angiotropin leads to defined changes of cell morphology that are dose dependent and reversible. In the presence of angiotropin, capillary endothelial cells rapidly form tubelike structures on gelatinized plates. This organizational state is not reached with aortic endothelial cells. The results indicate that the biological action of monocytic angiotropin is different from that of the angiogenic growth factors that stimulate the proliferation of endothelial cells and nonlymphoid mesenchymal cells and keep endothelial cells in the contact-inhibited epitheloid cell phenotype. We propose that angiotropin is directly involved in monocyte-induced angiogenesis.

Animals↗
PubMed1987

Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases.

Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns.

Amino Acid Sequence↗
PubMed1987

Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.

Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, have a molecular weight of about 18,400, cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.

Animals↗
PubMed1987

Regression of blood vessels precedes cartilage differentiation during chick limb development.

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.

Animals↗