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T F Deuel

Publications and source records attributed to T F Deuel.

At least 109 records · Page 6Linked to original sources
PubMed1984

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity.

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.

Animals↗
PubMed1984

Transforming protein of simian sarcoma virus stimulates autocrine growth of SSV-transformed cells through PDGF cell-surface receptors.

Simian sarcoma virus-transformed NIH 3T3 (SSV-NIH 3T3) and SSV-NRK cells secrete a potent growth-promoting activity identical with the platelet-derived growth factor (PDGF) in mitogenic assays. The secreted activity is blocked by anti-PDGF antisera and competes with 125I-PDGF for receptor binding, suggesting that the secreted protein is the transforming protein of SSV, p28v-sis, or its processed product. Secreted p28v-sis appears to stimulate autocrine cell growth of SSV-transformed cells because anti-PDGF antisera block 3H-thymidine incorporation into growing SSV-NIH 3T3 and SSV-NRK cells. SSV-transformed cells have reduced numbers of high-affinity 125I-PDGF receptors; PDGF/p28v-sis receptor was purified from SSV-NIH 3T3 cells and retained active protein tyrosine kinase activity stimulated by PDGF. The rate of tumor growth in athymic nude mice injected with SSV-transformed cells was compared with levels of secreted growth factor activity. The rate of tumor growth in nude mice correlated directly with levels of p28v-sis secreted by SSV-transformed cells.

Animals↗
PubMed1984

Specific covalent binding of platelet-derived growth factor to human plasma alpha 2-macroglobulin.

Attempts to measure the platelet-derived growth factor (PDGF) in human plasma resulted in the discovery of a specific plasma binding protein. The 125I-labeled PDGF (125I-PDGF)-plasma binding protein complex retained mitogenic activity but lost reactivity against rabbit anti-PDGF antiserum. Copurification of the plasma binding protein and alpha 2-macroglobulin (alpha 2M) in human plasma, the formation of a complex between 125I-PDGF and purified alpha 2M, and the comigration of the 125I-PDGF-plasma binding protein complex and the 125I-PDGF-alpha 2M complex in NaDodSO4/polyacrylamide gel electrophoresis and in pore-limiting polyacrylamide gel electrophoresis strongly suggested that alpha 2M is the plasma binding protein for 125I-PDGF. Immunoprecipitation of 125I-PDGF-alpha 2M and 125I-PDGF-plasma binding protein complexes by anti-human alpha 2M antiserum further established that alpha 2M and the plasma binding protein are the same molecule. Approximately 20% of 125I-PDGF is complexed by alpha 2M; further 125I-PDGF is complexed if the remaining 125I-PDGF is incubated with additional alpha 2M. Complex formation of 125I-PDGF with plasma or with alpha 2M was completely inhibited by 0.2 mM p-chloromercuric benzoate or 0.2 mM N-ethylmaleimide. The 125I-PDGF-alpha 2M complex or 125I-PDGF-plasma binding protein complex was not dissociated by 8 M urea, 1 M acetic acid, 0.1 M NaOH, or 1% NaDodSO4 but was dissociated by 2-mercaptoethanol, suggesting that the covalent binding of 125I-PDGF to alpha 2M occurs through a disulfide/sulfhydryl exchange reaction. The 125I-PDGF-alpha 2M complex (780,000 daltons) appears to contain two molecules of 125I-PDGF and two dimers of alpha 2M. The precise physiological role of the 125I-PDGF-alpha 2M interaction is unknown. alpha 2M may serve to limit PDGF released locally at sites of blood vessel injury. Alternatively, because of the nearly complete homology between the partial amino acid sequence of PDGF and the predicted amino acid sequence of the transforming protein of the simian sarcoma virus, p28sis, alpha 2M may play an important role in limiting the activity of a PDGF-like activity expressed by virus-transformed cells.

Humans↗
PubMed1984

Platelet-derived growth factor promotes polymorphonuclear leukocyte activation.

The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.

Calcium↗
PubMed1983

Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells.

The near identity of the partial amino acid sequence of human platelet-derived growth factor (PDGF) and that predicted for p28sis, the putative transforming protein of the simian sarcoma virus (SSV), suggests expression of a growth factor activity may be central for transformation by SSV. It is now reported that SSV-transformed cells but not control cells contain a growth factor activity that is identical to PDGF in immunoassay, in mitogenic dose response, and in specific mitogenic activity. The protein immunoprecipitated by antiserum to human PDGF has an apparent molecular weight of 20,000, identical to that of p20sis, the putative intracellular degradation product of p28sis. The results support the concept that expression of a PDGF-like molecule, which appears to be the product of the viral-sis gene, is responsible for the abnormal regulation of growth is SSV-transformed cells.

Amino Acid Sequence↗
PubMed1983

Platelet-derived growth factor stimulates the phosphorylation of ribosomal protein S6.

The human platelet derived-growth factor (PDGF) is both a potent mitogen and a strong chemoattractant protein for cells involved in inflammation and repair. In seeking mechanisms by which PDGF might initiate specific activities in target cells, it was found that highly purified PDGF stimulates the phosphorylation of an Mr approximately 33000 protein in confluent Swiss mouse 3T3 cells [Biochem. Biophys. Res. Commun. (1981) 103, 355-361]. The Mr approximately 33000 protein has now been recovered in polysomes by differential centrifugation and identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis.

Animals↗
PubMed1983

Platelet-derived growth factor elicits cyclic AMP accumulation in Swiss 3T3 cells: role of prostaglandin production.

Partially purified porcine PDGF or purified human PDGF in the presence of phosphodiesterase inhibitors caused marked accumulation of cAMP in Swiss 3T3 cells. The responses were time- and dose-dependent; half-maximal effect was obtained at 0.6 nM PDGF. Indomethacin prevented the increase of cAMP levels in a dose-dependent manner; half-maximal effect was obtained at about 10 nM. Addition of PDGF increased (at least 25-fold) the production of E-type prostaglandins; PGE reached a concentration in the medium of 26 ng/ml 1 hr after treatment with human PDGF. This concentration of PGE produced a similar level of cAMP to that found with PDGF, suggesting that the PDGF-induced increase in cAMP is mediated by E-type prostaglandins released in the culture medium. Increased cAMP levels promoted by PDGF acting through stimulation of E-type prostaglandin synthesis may contribute to signal the initiation of cell proliferation in 3T3 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Platelet-derived growth factor stimulates Na+/H+ exchange and induces cytoplasmic alkalinization in NR6 cells.

Stimulation of Na+/H+ exchange by growth factors has been implicated as a mechanism allowing quiescent cells to resume growth because of a predicted elevation of intracellular pH(pHi). We tested this prediction in NR6 cells by using a further development of our technique for pHi measurement, based on introduction of the fluorescent pH indicator 4',5'-dimethylfluorescein (pKa = 6.75) coupled to dextran into the cytoplasm. Addition of the potent mitogens platelet-derived growth factor (PDGF) or serum to NR6 cells stimulates an amiloride-sensitive 22Na+ uptake and causes an elevation of pHi. The PDGF-dependent pHi increase follows a lag period of approximately equal to 2 min, reaches a maximal level within 10 min (delta pHi approximately equal to 0.1 at an external pH of 7.18), and remains at this level for at least 1 hr. Serum addition initially produces a large elevation of pHi, which later declines to a level similar to that obtained with PDGF. The effects of PDGF and serum are partially additive (delta pHi approximately equal to 0.14). The magnitude of pHi elevation by PDGF decreases with increasing extracellular pH. Serum- and PDGF-dependent elevations of pHi are inhibited by amiloride and by eliminating Na+ from the medium. Under conditions in which Na+/H+ exchange is inhibited, PDGF and serum induce an initial cytoplasmic acidification that does not show a lag period. The results show that a single purified growth factor, as well as serum, can promote a sustained elevation of pHi by stimulating Na+/H+ exchange. The extent of pHi elevation may be modulated by the concomitant stimulation by the growth factor of a process generating H+ within the cell.

Amiloride↗
PubMed1983

Chemotactic activity of platelet alpha granule proteins for fibroblasts.

At sites of blood vessel injury, platelets release numerous substances that may have biological activities influencing cellular responses. In this study we examined separately the chemotactic activity for fibroblasts of three highly purified proteins obtained from platelet alpha granules: platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and beta-thromboglobulin (BTG). We observed that each of these proteins was strongly chemotactic for fibroblasts, with maximum chemotactic activity in each instance comparable to that observed with an optimal concentration of the control chemotactic protein, plasma fibronectin. Each protein was active at very low concentrations. The peak chemotactic activities of PF4, PDGF, and BTG occurred at 200 mg/ml, 30 ng/ml, and 6 ng/ml, respectively. Specificity of fibroblast chemotaxis to individual platelet proteins was provided by finding that anti-PF4 immunoglobulin blocked the chemotactic activity of PF4 without affecting the chemotactic activity of PDGF, while anti-PDGF immunoglobulin blocked the activity of PDGF but did not alter the capacity of PF4 to promote fibroblast chemotaxis. These results suggest that in vivo several alpha granule proteins released from platelets may affect wound healing by causing directed fibroblast migration.

Animals↗
PubMed1983

New synthesis of a platelet-specific protein: platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system.

The site of synthesis of platelet-specific proteins remains to be established. With the use of short-term megakaryocyte-enriched cultures, direct evidence was obtained to show that megakaryocytes synthesize the platelet-specific protein, platelet factor 4. A megakaryocyte-enriched fraction of rabbit bone marrow for culture was obtained by centrifugal elutriation and cultured with [3H]leucine. Newly synthesized 3H-platelet factor 4 was sought by copurification with added carrier rabbit platelet factor 4, using heparin agarose affinity chromatography and immunoprecipitation with specific goat anti-rabbit platelet factor 4 antisera. SDS PAGE of the washed immunoprecipitates demonstrated a [3H]leucine-containing peak which migrated identically with purified homogeneous rabbit platelet factor 4. A second, slightly larger molecular-weight protein was identified in the gels also, suggesting that rabbit platelet factor 4 may be synthesized as a larger molecular-weight precursor in rabbit megakaryocytes. These results provide direct evidence that the platelet-specific protein, platelet factor 4, is synthesized in rabbit megakaryocytes before it is packaged into alpha-granules for release in circulating platelets.

Animals↗
PubMed1983

Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein.

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.

Animals↗
PubMed1982

Proteoglycan carrier of human platelet factor 4. Isolation and characterization.

A large scale purification procedure for the human platelet factor 4 proteoglycan carrier molecule has been developed. A yield of 46% and a 33,000-fold purification have been achieved, using poly-L-lysine-Sepharose affinity column chromatography, PF4-agarose affinity column chromatography, and Bio-Gel A-0.5m gel filtration. The purified proteoglycan migrates as a single band during electrophoresis on cellulose acetate strips. A single symmetric peak was observed in sedimentation velocity analysis with an s value of 2.85. The molecular weight of the proteoglycan was determined to be 53,000 by sedimentation equilibrium. The purified proteoglycan contains 32% uronic acid, 31% galactosamine, 6.1% sulfate, and 9.9% protein. Aspartic acid, glutamic acid, leucine, glycine, and serine account for 55% of the total amino acids. The chondroitinase AC digest of the proteoglycan is sensitive to hydrolysis by chondro-4-sulfatase but not by chondro-6-sulfatase, indicating the presence of chondroitin 4-sulfate but not chondroitin 6-sulfate in the proteoglycan molecule. The interaction between this proteoglycan carrier of human PF4 and PF4 is strongly ionic strength-dependent. 0.3 M NaCl is required to dissociate the proteoglycan PF4 complex.

Amino Acids↗
PubMed1982

Platelet-derived growth factor. Specific binding to target cells.

The binding of the human platelet-derived growth factor (PDGF) to Swiss mouse 3T3 cells have been investigated. The binding is specific and reversible. The dissociation constant is approximately 0.7 x 10(-9) M with approximately 400,000 binding sites/cell. Two forms of PDGF, PDGF I (Mr = 31,000) and PDGF II (Mr = 28,000), previously identified (Deuel, T. F., Huang, J. S., Proffitt, R. T., Baenziger, J. U., Chang, D., and Kennedy, B. B. (1981) J. Biol. Chem. 256, 8896-8899 and Deuel, T. F., Huang. J. S., Proffitt, R. T., Chang, D., and Kennedy, B. B. (1981) J. Supramol. Cell Biochem. 5 (Suppl.), 128) bind equally well to 3T3 cells. Polylysine and histone, but not cytochrome c, partially inhibit the binding of PDGF to 3T3 cells. Protamine sulfate blocks binding in a competitive manner and is capable of displacing PDGF previously bound to the cell surface. EDTA influenced neither the binding of PDGF to the cell surface nor the displacement of cell-bound PDGF. At 37 degrees C, PDGF bound to the cell surface is lost and iodotyrosine is released free into the supernatant, with each process having a t 1/2 of approximately 90 min. The binding activity of the putative PDGF receptor is markedly reduced by previous incubation with PDGF, thereby apparently regulating its activity in a manner similar to epidermal growth factor.

Animals↗