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Biomedical subjects

A Wasteson

Publications and source records attributed to A Wasteson.

At least 37 records · Page 2Linked to original sources
PubMed1989

Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation.

Platelet-derived growth factor (PDGF) is known to inhibit collagen-induced platelet aggregation. Collagen-induced binding of 125I-PDGF to human washed platelets was therefore investigated. It was found 1) to be time-dependent, reaching a plateau at 20 degrees C after 30 min, 2) collagen concentration-dependent, 3) specifically inhibited by unlabeled PDGF, and 4) saturable. Scatchard plot analysis showed a single class of sites with 3000 +/- 450 molecules bound/cell and an apparent KD of 1.2 +/- 0.2 10(-8) M. The effects of PDGF on collagen-induced phosphoinositide breakdown and protein phosphorylation were also investigated. At 50 ng/ml PDGF, a concentration which completely inhibited collagen-induced aggregation, the breakdown of [32P]phosphatidylinositol 4,5-biphosphate (PIP2) and [32P]phosphatidylinositol 4-phosphate (PIP) was observed, but the subsequent replenishment of [32P]PIP2 was inhibited. The same PDGF concentration totally inhibited collagen-induced phosphatidic acid formation. PDGF also completely prevented phosphorylation of P43 and P20, as a result of protein kinase C activation consecutive to phosphoinositide metabolism. These results suggest that (i) a specific PDGF receptor can be induced by collagen, and (ii) PDGF can effect the early events of collagen-induced platelet activation by inhibiting PIP2 resynthesis and P43 and P20 phosphorylation. It is concluded that PDGF might be involved in a negative feed-back control of platelet activation.

Binding Sites↗
PubMed1989

Lateral diffusion of plasma membrane receptors labelled with either platelet-derived growth factor (PDGF) or wheat germ agglutinin (WGA) in human polymorphonuclear leukocytes and fibroblasts.

The aims of the present investigation were (a) to compare the lateral mobility of membrane receptors of human fibroblasts and polymorphonuclear leukocytes (PMNL) labelled with either platelet-derived growth factor (PDGF), or the lectin wheat germ agglutinin (WGA), and (b) to study effects of serum or PDGF on the mobility of these receptor molecules in human fibroblasts. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard (10%) or under serum-free conditions yielding "normal" and "starved" cells, respectively. The receptor mobility was studied in response to exposure to PDGF, or serum, in short time or prolonged incubations. Human polymorphonuclear leukocytes (PMNL) were adhered to microscope slides by clotting drops of blood. They were stained with rhodaminated PDGF or fluoresceinated WGA. The diffusion of labelled receptors was assessed with fluorescence recovery after photobleaching (FRAP). It was found that (a) fibroblasts grown at normal serum concentration had a lower diffusion coefficient (D = 3 x 10(-10) cm2 s-1) for the PDGF-receptor and a slightly lower mobile fraction (R = 60%) than starved cells (D = 5 x 10(-10) cm2 s-1 and R = 73%), (b) addition of serum to starved cells increased both D and R for the PDGF receptor to 12 x 10(-10) cm2 s-1 and 96%, respectively, (c) a similar pattern was obtained for WGA-labelled glycoconjugates indicating general membrane effects of serum-induced cell stimulation, and (d) in PMNL the PDGF receptor displayed motility characteristics (D = 3 - 4 x 10(-10) cm2 s-1 and R = 59%) similar to those in fibroblasts, possibly suggesting equivalent anchorage mechanisms in the membrane.

Cell Line↗
PubMed1989

Formation of chondroitin sulphate proteoglycan in cartilage regenerated from free perichondrial graft.

Perichondrium from rabbit auricular or rib cartilage was used as a free autogenous graft and transplanted either to the subcutaneous tissue of the back of the rabbit or to an experimental defect in the femur condyles. Outgrowth of new tissue, morphologically indistinguishable from cartilage, was observed after six weeks. Inorganic 35SO4, administered in vivo, was incorporated into the newly formed tissue. The labelled products were isolated, identified, and compared with those obtained from authentic cartilage of auricular, rib or joint surfaces. The products of newly formed cartilage were similar to those of authentic cartilage. The results support earlier morphological findings, indicating that perichondrium from rib cartilage has a better ability to regenerate than auricular perichondrium. The synovial environment seems to have a positive effect on the generation of cartilage.

Animals↗
PubMed1988

A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain.

Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.

Antigen-Antibody Complex↗
PubMed1988

Transforming growth factor (TGF beta) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding.

Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF beta were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. Moreover, electron microscopy excluded the presence of endothelial cells by the absence of Weibel-Palade bodies. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classes of sites were detected by Scatchard analysis with respectively 21,000 and 37,000 sites per cell, with a KD of 0.3 x 10(-10) M and KD of 0.5 x 10(-9) M. The stimulation of DNA synthesis by PDGF was quantified by [3H]thymidine incorporation. When PDGF was added alone at a concentration of 15 ng/ml, it induced a maximal DNA synthesis of 400%, which increased up to 900%, in the presence of platelet-poor plasma (PPP). On the other hand, PDGF-induced fibroblast proliferation was inhibited in a dose-dependent manner by TGF beta. This inhibition was related to a significantly decreased binding of 125I-labeled PDGF observed in the presence of TGF beta. Our results suggested that PDGF and TGF beta could modulate the growth of bone marrow fibroblasts.

Bone Marrow↗
PubMed1988

Platelet contribution to cancer cell growth and migration: the role of platelet growth factors.

Blood platelets contain several growth factors and inhibitors. The better known among them are named platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta), active as modulators of growth of normal mesenchymal and epithelial cells. Cancer cell growth in vitro seems to be independent of the effects of exogenous peptides, but dependent on the cell ability to release autocrine growth factors, similar to PDGF or TGF beta. In addition, platelet-associated growth factors and inhibitors, which are able to induce a fibrotic response in connective tissue cells, might also play a role to modulate the desmoplastic reaction surrounding the tumor.

Blood Platelets↗
PubMed1987

Megakaryocytes and myelofibrosis in gray platelet syndrome.

Studies have been carried out with cultured human megakaryocytes derived from Gray Platelet Syndrome blood and have demonstrated that MK-DGF and MK-F4 are present in the early stages of megakaryocyte maturation (day 5-6) but disappear in the late phase (day 9-11). In Gray platelets total mitogenic activity, PDGF and PF4 amounts were reduced while beta TG and PF4 plasma levels were slightly increased. These findings indicate a possible relationship between the loss of alpha granule contents from the megakaryocytes in the marrow and the myelofibrosis found in Gray platelet disorder and possibly associated in other diseases with platelet or megakaryocyte abnormalities.

Blood Platelet Disorders↗
PubMed1985

Raised serum hyaluronate levels in scleroderma: an effect of growth factor induced activation of connective tissue cells?

The circulating levels of hyaluronate were determined in 36 patients with scleroderma and in 36 control subjects matched for age and sex. The mean serum hyaluronate concentration in patients with progressive systemic sclerosis (n = 25) was 131 +/- 67 (SD) microgram/l and significantly greater (p less than 0.001) than that of the controls (mean level 49 +/- 21 (SD) microgram/l). Hyaluronate levels in patients with localised scleroderma (n = 4) were 141 +/- 47 (SEM) microgram/l and in patients with scleroderma-associated overlap syndromes (n = 7) 202 +/- 54 (SEM) microgram/l. The increase in serum hyaluronate probably reflected an enhanced synthesis or outflow of hyaluronate from the connective tissue, or both; it could not be explained by affection of the liver, which is the catabolic site of hyaluronate. The hyaluronate values were not related to certain serological indicators of inflammatory activity or to the extent of the skin lesions or the severity of internal organ manifestations. A positive correlation was noted between circulating platelet counts and hyaluronate levels (p less than 0.001). Plasma beta-thromboglobulin was measured in 15 of the patients with systemic sclerosis and found to correlate positively with platelet counts. Raised levels of beta-thromboglobulin were associated with the highest hyaluronate values. Platelet-derived growth factor, which stimulates connective tissue cells and is stored in the alpha-granules of platelets together with beta-thromboglobulin, was shown to enhance hyaluronate synthesis in fibroblast cultures. The results suggest an involvement in scleroderma of connective tissue activating substances released from platelets.

Cells, Cultured↗
PubMed1985

Platelet-derived growth factor: mechanism of action and relation to oncogenes.

Recent studies of platelet-derived growth factor (PDGF) have revealed several structural and functional similarities between this growth factor or components linked to its mechanism of action and certain oncogene products: PDGF itself has a structural homology with the transforming protein of simian sarcoma virus, the PDGF receptor has a functional homology (tyrosine kinase activity) with a family of oncogene products, and PDGF induces the expression of the cellular counterparts of myc and fos. In addition, several tumour cell lines have been found to produce PDGF-like growth factors, which may cause autocrine stimulation of growth. We interpret these findings as indicating that regulatory components along the PDGF-dependent mitogenic pathway may have oncogenic properties if they are inappropriately expressed or activated.

Cell Line↗
PubMed1984

A chondroitin sulphate proteoglycan from human cultured glial and glioma cells. Structural and functional properties.

A chondroitin sulphate proteoglycan capable of forming large aggregates with hyaluronic acid was identified in cultures of human glial and glioma cells. The glial- cell- and glioma-cell-derived products were mutually indistinguishable and had some basic properties in common with the analogous chondroitin sulphate proteoglycan of cartilage: hydrodynamic size, dependence on a minimal size of hyaluronic acid for recognition, stabilization of aggregates by link protein, and precipitability with antibodies raised against bovine cartilage chondroitin sulphate proteoglycan. However, they differed in some aspects: lower buoyant density, larger, but fewer, chondroitin sulphate side chains, presence of iduronic acid-containing repeating units, and absence (less than 1%) of keratan sulphate. Apparently the major difference between glial/glioma and cartilage chondroitin sulphate proteoglycans relates to the glycan rather than to the protein moiety of the molecule.

Cells, Cultured↗
PubMed1984

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.

Amino Acid Sequence↗
PubMed1984

A glioma-derived analog to platelet-derived growth factor: demonstration of receptor competing activity and immunological crossreactivity.

A human clonal glioma cell line, U-343 MGa Cl 2, cultured under serum-free conditions, was found to release a factor that competed with 125I-labeled platelet-derived growth factor (125I-PDGF) for binding to human foreskin fibroblasts. The concentration of competing activity in conditioned medium was equal to 20-30 ng of PDGF per ml. The PDGF receptor competing activity had an elution position on Sephadex G-200 close to that of tracer PDGF. The same fractions in the chromatogram also contained growth-promoting activity and material active in a PDGF radioimmunoassay. Incubation of partially purified, 125I-labeled glioma factor with fibroblasts, or rabbit anti-PDGF serum, led to the selective binding of a component with an estimated Mr of 31,000, as shown by NaDodSO4/gel electrophoresis under nonreducing conditions. After reduction this component migrated as a Mr 18,000 protein. Thus, the behavior in NaDodSO4/gel electrophoresis was similar to that of PDGF. Furthermore, incubation of partially purified glioma factor with immobilized PDGF antibodies markedly decreased the amount of PDGF receptor competing activity remaining in the supernatant. These results suggest that the factor produced by glioma cells has structural, immunological, and functional resemblance to PDGF. We previously reported that a human osteosarcoma cell line produces a PDGF-like molecule with growth-promoting activity. Taken together with the recent finding that PDGF is homologous to the transforming gene product of simian sarcoma virus, our present data give additional support for the idea that an autocrine activation of the PDGF receptor may be operational in the growth of human tumors of mesenchymal or glial origin.

Binding, Competitive↗
PubMed1983

Synthesis of a PDGF-like growth factor in human glioma and sarcoma cells suggests the expression of the cellular homologue to the transforming protein of simian sarcoma virus.

Several human normal and neoplastic cell lines were screened for production of PDGF receptor competing activity. Conditioned medium from two sarcomas and one glioma blocked 125I-PDGF binding to human foreskin fibroblasts in a dose-dependent manner. In each case this effect was abolished when the conditioned medium was pretreated with PDGF-antiserum, indicating that the receptor competing activity was immunologically related to PDGF. Direct evidence for de novo synthesis of a PDGF-like component in the cultures was afforded by 35S-cysteine labeling of the three cell lines, followed by immunoprecipitation with PDGF antiserum. This resulted in the specific precipitation of a 31,000 molecular weight labeled protein, which upon reduction was split into two polypeptides of molecular weights 17,000 and 16,500. The significance of these findings in view of the recently discovered structure homology between PDGF and the transforming gene product of simian sarcoma virus, p28sis, is discussed.

Binding, Competitive↗
PubMed1983

Surface binding and internalization of platelet-derived growth factor in human fibroblasts.

Surface binding and uptake of platelet-derived growth factor (PDGF) in human fibroblasts cultivated in vitro were studied by quantitative electron microscopic autoradiography using 125I-labeled PDGF and by indirect immunofluorescence using PDGF antibodies. After 120 min at 12 degrees C PDGF was found preferentially in coated regions of the plasma membrane. Warming the cells to 37 degrees C initiated rapid ingestion of the factor via small vesicles, usually lacking a membrane coat. After 10 min PDGF started to appear in lysosomes, and it showed maximal concentration within these organelles after 30 min. There were also signs of passage of PDGF through the Golgi complex, but only after 60 min. Treatment of the cells with chloroquine, a weak base that inhibits intralysosomal degradation, prevented disappearance of tracer from the lysosomes. The observations indicate that PDGF was internalized via coated regions of the plasma membrane and carried to lysosomes for degradation. Subsequent appearance of tracer within the Golgi complex could reflect receptor-ligand complexes that escaped degradation and were recirculated back to the cell surface.

Autoradiography↗