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Biomedical subjects

D T Connolly

Publications and source records attributed to D T Connolly.

33 records · Page 2Linked to original sources
PubMed1991

Free bone graft reconstruction of irradiated facial tissue: experimental effects of basic fibroblast growth factor stimulation.

A study was undertaken to evaluate the potential utility of basic fibroblast growth factor in the induction of angiogenesis and osseous healing in bone previously exposed to high doses of irradiation. Thirty New Zealand rabbits were evaluated by introducing basic fibroblast growth factor into irradiated mandibular resection sites either prior to or simultaneous with reconstruction by corticocancellous autografts harvested from the ilium. The fate of the free bone grafts was then evaluated at 90 days postoperatively by microangiographic, histologic, and fluorochrome bone-labeling techniques. Sequestration, necrosis, and failure to heal to recipient osseous margins was observed both clinically and histologically in all nontreated irradiated graft sites as well as those receiving simultaneous angiogenic stimulation at the time of graft placement. No fluorescent activity was seen in these graft groups. In the recipient sites pretreated with basic fibroblast growth factor prior to placement of the graft, healing and reestablishment of mandibular contour occurred in nearly 50 percent of the animals. Active bone formation was evident at cortical margins adjacent to the recipient sites but was absent in the more central cancellous regions of the grafts.

Animals↗
PubMed1990

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration.

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.

Amino Acid Sequence↗
PubMed1990

Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor.

Rodent and human tumor cell lines secrete a potent vascular permeability factor (VPF) which causes a rapid and substantial increase in microvascular permeability to plasma proteins without causing mast cell degranulation, or endothelial cell damage or without exciting an inflammatory cell infiltrate [D. R. Senger, S. J. Galli, A. M. Dvorak, C. A. Perruzzi, V. S. Harvey, and H. F. Dvorak. Science (Wash. DC), 219: 983-985, 1983; D. R. Senger, C. A. Perruzzi, J. Feder, and H.F. Dvorak. Cancer Res., 46: 5629-5632, 1986]. VPF now has been purified to homogeneity from guinea pig tumor cell culture medium; it is a Mr 34,000-43,000 protein, and a NH2-terminal amino acid sequence has been derived. A synthetic peptide corresponding to amino acid residues 1-24 of the native protein was used to raise rabbit antibodies which bind all of the vessel permeability-increasing activity secreted by guinea pig tumor cells and which stain purified VPF on immunoblots. These findings establish that this NH2-terminal amino acid sequence was derived from the permeability factor. Homology searches found no identity or close similarity between VPF NH2-terminal sequence and database sequences, indicating that VPF is distinct from other proteins for which sequence data are available. In particular, no sequence similarity was found between tumor-secreted VPF and other mediators of increased vessel permeability including plasma and glandular kallikreins.

Amino Acid Sequence↗
PubMed1989

Vascular permeability factor, an endothelial cell mitogen related to PDGF.

Vascular permeability factor (VPF) is a 40-kilodalton disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth, and angiogenesis. These properties suggest that the expression of VPF by tumor cells could contribute to the increased neovascularization and vessel permeability that are associated with tumor vasculature. The cDNA sequence of VPF from human U937 cells was shown to code for a 189-amino acid polypeptide that is similar in structure to the B chain of platelet-derived growth factor (PDGF-B) and other PDGF-B-related proteins. The overall identity with PDGF-B is 18%. However, all eight of the cysteines in PDGF-B were found to be conserved in human VPF, an indication that the folding of the two proteins is probably similar. Clusters of basic amino acids in the COOH-terminal halves of human VPF and PDGF-B are also prevalent. Thus, VPF appears to be related to the PDGF/v-sis family of proteins.

Amino Acid Sequence↗
PubMed1989

Human vascular permeability factor. Isolation from U937 cells.

Human vascular permeability factor (hVPF) is a glycoprotein that promotes fluid and protein leakage from blood vessels. The function of hVPF is at present unknown, but the potent bioactivities of this protein suggest that it could act during inflammation, wound healing, and tumor angiogenesis. hVPF was purified from serum-free conditioned medium of the human histiocytic lymphoma cell line U937 as a disulfide-linked dimeric 40-kDa protein that promoted dermal blood vessel leakage in guinea pigs at a dose of 20 ng (3 x 10(-9) M) and promoted in vitro endothelial cell growth at concentrations as low as 50 PM. Multiple forms of hVPF with apparent pI values greater than 7.5 were resolved using pH gradient electrophoresis. Antibodies against guinea pig vascular permeability factor were found to cross-react with hVPF. The N-terminal amino acid sequence of hVPF was similar to, but not identical with, the N-terminal sequence of guinea pig vascular permeability factor.

Amino Acid Sequence↗
PubMed1989

Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis.

Amino Acid Sequence↗
PubMed1988

Enhancement of angiogenesis by bFGF in mandibular bone graft healing in the rabbit.

The effectiveness of basic fibroblast growth factor (bFGF) as an in vivo angiogenic factor was evaluated in autogeneic bone grafts to the rabbit mandible. Block cortical grafts harvested from the ilium were implanted into sites in the mandibular ramus or body. The basic fibroblast growth factor was continuously introduced over a period of 14 days through subcutaneous osmotic pumps. Increased vascularity, as assessed by vessel number and depth of penetration into the grafts, was noted at 10 days postoperatively in the bFGF stimulated side as compared to contralateral control grafts. At the fourteenth postoperative day, bFGF administration was discontinued and a decrease in angiogenesis was noted over the ensuing 2 weeks so that after 1 postoperative month, little difference could be detected between the stimulated and nonstimulated grafts. Assessment of osseous healing at the 1 month postoperative interval using triple fluorochrome labeling did not reveal evidence of accelerated osteogenesis on the previously stimulated side.

Angiography↗
PubMed1987

Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells.

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.

Animals↗
PubMed1986

Determination of the number of endothelial cells in culture using an acid phosphatase assay.

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.

Acid Phosphatase↗
PubMed1983

Binding and endocytosis of glycoproteins and neoglycoproteins by isolated rabbit hepatocytes.

Rabbit hepatocytes were isolated by a collagenase perfusion technique, and used to study the binding and endocytosis of the glycoprotein, asialo-orosomucoid, and the neoglycoprotein, Gal39-bovine serum albumin. Both of these proteins contain exposed galactosyl residues, and were avidly bound by the lectin on the hepatic parenchymal cell surface. Steady state and kinetic experiments performed at 2 degrees C and at 37 degrees C revealed the presence of two apparent classes of binding sites totalling 4.7 X 10(5) sites/cell at 2 degrees C, and 6.3 X 10(5) sites/cell at 37 degrees C. At 37 degrees C, both classes of sites participated in internalization of bound ligand. The cells were capable of internalizing about 60 000 molecules/min per cell. The process appeared to be first-order, with a rate constant k = 0.098 min-1 and t1/2 = 7.1 +/- 0.6 min. Binding could be inhibited by galactose-containing compounds, EGTA, and by anti-(hepatic lectin) immunoglobulin G. The inhibition by antibody appeared to be reversible upon removal of antibody-containing medium.

Animals↗
PubMed1982

Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts.

Epidermal growth factor (EGF) receptors extracted with Triton X-100 from human skin fibroblasts and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin fibroblasts and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = 16 h) than that (t1/2 = 9 h) of fibroblasts. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from 16 to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.

Carcinoma, Squamous Cell↗
PubMed1982

Binding and endocytosis of cluster glycosides by rabbit hepatocytes. Evidence for a short-circuit pathway that does not lead to degradation.

Synthetic cluster glycosides containing either one, two, or three galactosyl or lactosyl residues per ligand were used to test the effect of carbohydrate clustering on binding by the rabbit hepatic Gal/GalNAc-binding lectin using either isolated rabbit hepatocytes or the solubilized, affinity-purified lectin. The tris- and bis-glycosides were superior to the mono-glycosides for inhibition of 125I-asialoorosomucoid binding to rabbit hepatocytes at 0 degrees C. The concentrations of the tris-glycosides required for 50% inhibition of 125I-asialoorosomucoid binding (4-8 microM) to hepatocytes were 50-100 times lower than the concentrations of the corresponding mono-glycosides required for 50% inhibition (400-500 microM). The isolated lectin, however, did not effectively discriminate between the mono-, bis-, and tris-glycosides, possibly indicating an organizational difference between the lectin in the cell membrane and the isolated lectin. When the cluster glycosides were labeled with 125I-tyrosine, it was shown that the tris- and bis-galactosides were bound to the hepatocytes at 37 degrees C, and that binding was followed by a step that led to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid resistance, probably internalization. The process could be specifically inhibited by the neoglycoprotein Gal44-AI-bovine serum albumin, or by IgG specific for the hepatic lectin, but not by preimmune IgG. Internalization of the cluster glycosides did not lead to accumulation of ligand inside the cell, nor to degradation. Instead, the ligands were quickly released from the cells.

Animals↗
PubMed1981

Steady state and kinetic analysis of the binding of asialoorosomucoid to the isolated rabbit hepatic lectin.

The binding of 125I-asialoorosomucoid and the isolated rabbit hepatic lectin were studied at 0 degrees C. The steady state binding data were analyzed both by a direct curve-fitting procedure that utilizes nonlinear least squares regression analysis, and by conventional Scatchard plot analysis. Two classes of binding sites, present in approximately equal concentrations, were detected by both analytical procedures. The total amount of 125I-asialoorosomucoid bound was between 0.2 and 1.0 mol/mol of lectin for each class of lectin binding sites, if the molecular weight of the lectin is between 100,000 and 500,000. The estimated apparent equilibrium dissociation constants were Kapp = 0.87 nM and 1100 nM using the direct curve-fitting method and 0.63 nM and 43 nM from Scatchard plot analysis. Analysis of the forward binding reaction yielded apparent rate constants k1 = 6.8 X 10(6) M-1 and k-1 = 1.5 X 10(-2) min-1, and, therefore, Kapp = 2.1 nM. (Kapp is the apparent dissociation constant of the complex between asialoorosomucoid and hepatic lectin, K1 is the bimolecular rate constant for association of asialoorosomucoid and hepatic lectin, and K-1 is the rate constant for dissociation of the asialoorosomucoid-hepatic lectin complex.) In contrast, the reverse reaction was too slow in the presence of excess unlabeled asialoorosomucoid, and too fast in the presence of methyl beta-D-galactopyranoside to be accommodated by the simple bimolecular reaction model.

Animals↗