Platelet-derived growth factor isoforms AA, AB, and BB differentially activate poly r(I):r(C)-induced genes in human fibroblast FS4 cells.

Polyribocytidylic-polyriboinosinic acid [poly r(I):r(C)]-inducible genes were isolated by a differential screening procedure from a human fibroblast cell (FS-4) cDNA bank. Among yet unidentified genes (gene 274), one codes for a protein with multiple finger motifs and has previously been detected in endothelial cells after tumor necrosis factor-alpha (TNF-alpha) treatment (A20; Opipari et al., 1990), the second one codes for a variant of the I kappa B family (Haskill et al., 1991), and a third one for the Ca2+ ATPase (isoform 1). Platelet-derived growth factor (PDGF) isoforms (AA, AB, and BB) stimulated the expression of these immediate-early genes. But the extent of the respective induction correlated neither with the number of the two receptors alpha or beta nor with the level of PDGF-stimulated receptor autophosphorylation on tyrosine. Although alpha-receptors were less abundant than beta-receptors (12,500 binding sites were estimated for PDGF-AA, KD 0.03 nM; 20,000 for PDGF-AB, KD 0.03 nM; 35,000 for PDGF-BB KD 0.16 nM) and tyrosine phosphorylation induced by PDGF-AA was significantly less than that evoked by PDGF-BB, some of the investigated genes were more strongly induced by PDGF-AA. We discuss how the differences in the biological potency of the PDGF isoforms may reside in different functions of the two receptors by activation of alternative signaling pathways.     

Differential effects of PDGF and PDGF-BB on vascular smooth muscle cells

Several biological effects of recombinant PDGF-BB and PDGF obtained from human platelets were examined with vascular smooth muscle cells. Although PDGF and PDGF-BB were equally potent mitogens for these cells, 5 fold higher levels of PDGF were required to displace 125I-PDGF-BB binding than PDGF-BB itself. Higher concentrations of PDGF relative to PDGF-BB were also required to stimulate the phosphorylation of a 163K protein in membrane preparations. PDGF-BB, but not PDGF, treatment of intact cells resulted in the phosphorylation on tyrosine residues of 168, 53, 48, and 45K proteins. The data suggest that PDGF and PDGF-BB stimulate smooth muscle cell mitogenesis by different mechanisms.     

Divergence of binding, signaling, and biological responses to recombinant human hybrid IFN.

Three human IFN-alpha hybrids, HY-1 [IFN-alpha21a(1-75)/alpha2c(76-165)], HY-2 [IFN-alpha21a(1-95)/alpha2c(96-165)], and HY-3 [IFN-alpha2c(1-95)/alpha21a(96-166)], were constructed, cloned, and expressed. The hybrids had comparable specific antiviral activities on Madin-Darby bovine kidney (MDBK) cells but exhibited very different antiproliferative and binding properties on human Daudi and WISH cells and primary human lymphocytes. Our data suggest that a portion of the N-terminal region of the molecule is important for interaction with components involved in binding of IFN-alpha2b while the C-terminal portion of IFN is critical for antiproliferative activity. A domain affecting the antiproliferative activity was found within the C-terminal region from amino acid residues 75-166. The signal transduction properties of HY-2 and HY-3 were evaluated by EMSA and RNase protection assays. Both HY-2 and HY-3 induced activation of STAT1 and 2. However, HY-2 exhibited essentially no antiproliferative effects at concentrations that activated STAT1 and 2. Additionally, at concentrations where no antiproliferative activity was seen, HY-2 induced a variety of IFN-responsive genes to the same degree as HY-3. RNase protection assays also indicate that, at concentrations where no antiproliferative activity was seen for HY-2, this construct retained the ability to induce a variety of IFN-inducible genes. These data suggest that the antiproliferative response may not be solely directed by the activation of the STAT1 and STAT2 pathway in the cells tested.     

重组截短型人源结缔组织生长因子在大肠杆菌中的表达

用RT－PCR法从人胎盘组织中克隆出人源结缔组织生长因子（h-CTGF)cDNA序列867bp,将此cDNA亚克隆至表达载体pET-9a,重组质粒转化BL21（DE3）pLysS,诱导出N端缺失61个氨基酸残基的截短型rh－CTGF,表达量占总菌体蛋白的7％,主要以不溶性包涵体形式存在,采用离心、洗涤和凝胶过滤分离纯化后,行活性检测表明截短型rt-CTGF无刺激增殖活性,并对用原核表达rt-CTGF作了讨论,为制备抗体、CTGF表达调控和功能研究打下了基础。     

The relation between connective tissue cells and intercellular substances, including basement membranes.

Basement membranes are distributed widely in the body forming an extracellular matrix for epithelial and endothelial cells. The collagenous and glycoprotein constituents of basement membranes are synthesized by these two cell types. Disturbance of the interactions between basement membranes and their associated epithelial and endothelial cells can lead to the pathological changes seen in diseases involving basement membranes. These changes are illustrated here by reference to glomerulonephritis induced by the deposition of immune complexes in the glomerulus of the kidney, and chronic inflammatory changes occurring in the lung after inhalation of asbestos. In these diseases basement membrane changes can occur in several ways. Hydrolytic enzymes released from inflammatory cells degrade basement membranes while other constituents by epithelial and endothelial cells. Alternatively the physical separation of epithelial and endothelial cells from their basement membrances by space-occupying substances such as immune complexes can interfere with feedback mechanisms leading to synthesis of basement membrane constituents and cell proliferation. Studies of these pathological changes at a cellular level should shed new light on the ways in which cells interact with their pericellular environment.     

Differential regulation of biglycan and decorin synthesis by connective tissue growth factor in cultured vascular endothelial cells

It is possible that connective tissue growth factor (CTGF) serves as either an independent regulator or a downstream effector of transforming growth factor-beta (TGF-beta) on the proteoglycan synthesis in vascular endothelial cells. Since TGF-beta regulates endothelial proteoglycan synthesis in a cell density-dependent manner, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(35)S]sulfate or (35)S-labeled amino acids in the presence of CTGF, and the labeled proteoglycans were characterized by biochemical techniques. The results indicate that CTGF suppresses the synthesis of biglycan but newly induced that of decorin in the cells when the cell density is low; in addition, no change was observed in the hydrodynamic size and the glycosaminoglycan chain length of these two small chondroitin/dermatan sulfate proteoglycans. The regulation of endothelial proteoglycan synthesis by CTGF is completely different from that by TGF-beta, suggesting that CTGF is not a downstream effector of TGF-beta but an independent regulator in vascular endothelial cells with respect to the proteoglycan synthesis.     

The synthesis of connective tissue protein in smooth muscle cells.

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.     

Differential response of embryonic cells to culture on tissue matrices.

Cell responses to different natural substrates have been followed by scanning microscopy in order to evaluate the role of these substrates in morphogenesis. Matrix has been isolated then repopulated with suspensions of embryonic cells from chick skin, spinal ganglia, duodenal epithelium and heart. In some cases outgrowth from amphibian embryonic tissue was used. Basal lamina of the Xenopus tail may be exposed by freezing and thawing the tissue, or by EDTA treatment. The underlying lamella of orthogonally oriented collagen fibers may be exposed by use of trypsin or hyaluronidase. Trypsin causes more clumping of collagen fibers and a coarser texture of the matrix. On trypsin isolated basement lamella, nerve cell processes grow out on the surface and show no strong tendency to penetrate the lamella while skin mesenchymal cells commonly burrow among the collagen plies. Epithelial cells remain on the surface. On the basal lamina mesenchymal cells ruffle in early stages of culture, then flatten. Epithelial cells flatten rapidly on the lamina. These differences in cell response are in some cases closely related to cell behavior in vivo and suggest that cells show a selective response to the chemical composition of the substrate as well as to its physical conformation.     

Granular cells in the connective tissue of Helix pomatia L. (Gastropoda,Pulmonata)

Summary Granular cells (cells crowded with colourless granules staining with paraldehyde fuchsin according to Gomori-Gabe and not containing calcium) are independent cells in the connective tissue of Helix pomatia. Histochemical data suggest that the granules are rich in sulfhydryl-containing proteins, but lack biogenic monoamines. Electron microscopic investigations confirm the supposed secretory activity of the granular cells. Secretory proteins are presumed to be synthetized in the endoplasmic reticulum and condensed in the Golgi apparatus giving rise to the granules. Extrusion occurs by exocytosis.Electrophoresis of homogenates, prepared from tissues containing numerous granular cells, results in the separation and identification of a secretory protein from the granular cells. An electrophoretically homologous protein is recognized in the hemolymph, but in very small quantities.Our findings and the work of others suggest the involvement of granular cells in neuroendocrine events.The author is indebted to Prof. Dr. D. Kuhlmann for suggesting the problem and for his valuable criticism during the investigation. I would like to thank Mr. J.N. Howell, who helped with the English.Part of this work has been supported by the Stiftung Volkswagenwerk and by the Deutsche Forschungsgemeinschaft     

Heparin amplifies platelet-derived growth factor (PDGF)- BB-induced PDGF alpha -receptor but not PDGF beta -receptor tyrosine phosphorylation in heparan sulfate-deficient cells. Effects on signal transduction and biological responses

Platelet-derived growth factor (PDGF) induces mitogenic and migratory responses in a wide variety of cells, by activating specific receptor tyrosine kinases denoted the PDGF alpha- and beta-receptors. Different PDGF isoforms bind in a distinct manner to glycosaminoglycans, particularly heparan sulfate. In the present study, we show potentiation by exogenous heparin of PDGF-BB-induced PDGF alpha-receptor tyrosine phosphorylation in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells. This effect was not seen for PDGF-AA treatment, and heparin lacked a potentiating effect on PDGF-BB stimulation of the PDGF beta-receptor. Heparin did not affect the affinity of PDGF-BB binding for the PDGF receptors on CHO 677 cells. The PDGF-BB-stimulated PDGF alpha-receptor phosphorylation was enhanced in a dose-dependent fashion by heparin at low concentration. The effect was modulated by 2-O- and 6-O-desulfation of the polysaccharide. Maximal induction of PDGF alpha-receptor tyrosine phosphorylation (6-fold) in CHO 677 cells was achieved by treatment with a heparin decasaccharide, but shorter oligosaccharides consisting of four or more monosaccharide units were also able to augment PDGF alpha-receptor phosphorylation, albeit at higher concentrations. Heparin potentiated PDGF-BB-induced activation of mitogen-activated protein kinase and protein kinase B (Akt) and allowed increased chemotaxis of the CHO 677 cells toward PDGF-BB. In conclusion, heparin modulates PDGF-BB-induced PDGF alpha-receptor phosphorylation and downstream signaling, with consequences for cellular responsiveness to the growth factor.     

The binding,transport and fate of aluminium in biological cells

Aluminium is the most abundant metal in the Earth's crust and yet, paradoxically, it has no known biological function. Aluminium is biochemically reactive, it is simply that it is not required for any essential process in extant biota. There is evidence neither of element-specific nor evolutionarily conserved aluminium biochemistry. This means that there are no ligands or chaperones which are specific to its transport, there are no transporters or channels to selectively facilitate its passage across membranes, there are no intracellular storage proteins to aid its cellular homeostasis and there are no pathways which evolved to enable the metabolism and excretion of aluminium. Of course, aluminium is found in every compartment of every cell of every organism, from virus through to Man. Herein we have investigated each of the ‘silent’ pathways and metabolic events which together constitute a form of aluminium homeostasis in biota, identifying and evaluating as far as is possible what is known and, equally importantly, what is unknown about its uptake, transport, storage and excretion.