Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.     

Synthesis of platelet-derived growth factor by cells of splenic red pulp in normal rats

A population of cells in the spleens of normal rats was found to contain platelet-derived growth factor (PDGF) B chain mRNA. These cells were found predominantly in the red pulp and nuclear morphology of some was consistent with that of macrophages. Similar cells were also shown by immunocytochemical staining to contain PDGF-AB/BB. These PDGF-positive cells were also found almost exclusively in the red pulp. It has been suggested by others that PDGF plays an important role in the function of the lymphohemopoietic microenvironment.     

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571-treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation.     

Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is used to treat full-thickness diabetic ulcers and is being investigated for use in other chronic ulcers, non-healing wounds, and periodontal defects. A simple, novel method for expression and purification of rhPDGF-BB from Escherichia coli is now described. This method produces the dimeric protein in high yield (10–12 mg/g wet cell mass) and with a purity >95%. rhPDGF-BB was exclusively found in inclusion bodies (IBs) representing approx. 30% of the total cell proteins. The IBs were extracted and the monomer purified by RP-HPLC. The purified rhPDGF-B monomer was then refolded using Tris buffer and subsequently dimerized to produce biologically active rhPDGF-BB. This product was composed of two polypeptide chains, each approx. 12 kDa. The final product exhibited specific activity in a fibroblast proliferation assay indistinguishable from that of the WHO reference standard.     

Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor

In the present study, we investigated the effect of leptin on proliferation of hepatic stellate cells (HSCs) in vitro. Proliferation of 3-day cultured rat HSCs was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the nuclei. The percentages of BrdU-positive cells were increased in the presence of PDGF-BB (5 ng/ml) for 8h as expected. Co-incubation with leptin (10-100 nM) potentiates this PDGF-dependent increase in BrdU positive cells in a dose-dependent manner. Messenger RNA for PDGF receptor alpha and beta subunits was increased almost 2- to 3-fold by incubation with leptin for 6h. Further, pre-incubation with leptin for 6h enhanced PDGF-induced increases in phospho-p44/42 MAP kinase and phospho-Akt levels in a dose-dependent manner. In the same condition, however, leptin per se did not increase phospho-STAT 3 and phospho-p44/42 MAP kinase levels. Instead, leptin increased phospho-Akt levels in HSCs within 30 min, suggesting that the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in the mechanism by which leptin accelerates the proliferation of HSCs. In conclusion, the present study clearly indicated that leptin potentiates PDGF-dependent proliferative responses of HSCs in vitro.     

Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor

Summary The weak base chloroquine and the Na⁺/H⁺ ionophore monensin were used to study the role of lysosomes in the induction of DNA synthesis by platelet-derived growth factor (PDGF) in rat arterial smooth muscle cells cultivated in vitro. The results show that PDGF initiates DNA synthesis in a defined, serum-free medium. This indicates that a single factor may control, directly or indirectly, the transition from the G0 to the G1 phase, the progress through the G1 phase, and the entrance into the S phase of the cell cycle. It is further demonstrated that PDGF has to be present throughout most of the prereplicative period (12–16 h) to induce DNA synthesis in the maximum number of cells, suggesting that one or more processes need to be stimulated continually or successively to push the cell into the S phase. Chloroquine and monensin inhibit induction of DNA replication by PDGF, with maximum effect at 50 M and 5 M, respectively. To be fully active, the drugs have to be added within 4–8 h after the growth factor, but a partial inhibition persists if they are added at any time during the prereplicative period. Both drugs reduce PDGF-stimulated RNA and protein synthesis, and suppress degradation of [³H]leucine-labeled cellular protein and [¹²⁵I]-labeled PDGF. Fine-structurally, they give rise to an accumulation of lysosomes or prelysosomal vacuoles with inclusions of incompletely degraded material. These findings suggest that the mitogenic effect of PDGF is dependent on a normal function of lysosomes during the prereplicative phase, especially its first half (0–8 h).     

Suramin inhibits binding and degradation of platelet-derived growth factor in arterial smooth muscle cells but does not interfere with autocrine stimulation of DNA synthesis

Summary During in vitro culture arterial smooth muscle cells of adult rats are able to produce a platelet-derived growth factor (PDGF)-like protein and to promote their own growth in an autocrine manner. Here, this process has been studied using suramin, a polyanionic drug that has been reported to interfere with the cellular binding of several growth factors. Our results indicate that suramin speeds up the transition of the cells from a contractile to a synthetic phenotype early in primary culture. It inhibits the binding of PDGF to the cells, displaces PDGF bound to the cell surface, and slows down the degradation of PDGF internalized by the cells. It reduces the specific activities of the lysosomal enzymes acid phosphatase, -N-ace-tylglucosaminidase and -glucuronidase, and gives rise to an accumulation of lysosomes with myelin-like inlcusions. It blocks PDGF- and serum-induced DNA synthesis and cellular proliferation in secondary cultures, but lacks a distinct inhibitory effect on DNA synthesis in primary cultures under serum-free conditions. The results suggest that the PDGF-like protein produced by the smooth muscle cells under the latter conditions may bind to its receptor and exert its autocrine effect intracellularly, without prior release into the pericellular space.     

重组人可溶性PDGFRβ/Fc在昆虫细胞Sf9中的表达

【目的】利用昆虫细胞Bac-to-Bac杆状病毒表达系统表达血小板源性生长因子受体β (PDGFRβ)链膜外区与人IgG Fc片段的可溶性受体融合蛋白sPDGFRβ/Fc,并检测重组蛋白的特异性和生物活性。【方法】采用Bac-to-Bac系统,构建重组转移质粒pFastbac-sPDGFRβ/Fc,转化到含穿梭载体Bacmid的感受态细胞DH10Bac中,使目的基因与杆状病毒基因组DNA发生位点特异性重组,获得重组病毒DNA,将其通过脂质体转染昆虫细胞Sf9获得重组病毒。将该重组病毒感染Sf9无血清细胞系,在Sf9细胞中表达sPDGFRβ/Fc,对表达产物进行Western blotting检测和Protein A亲合层析纯化,并进一步通过MTT法检测获得的重组蛋白生物学活性。【结果】重组病毒感染Sf9细胞后,经Western blotting分析,能检测到一条分子量约为97 kDa的特异性条带,与目的蛋白大小相符。通过Protein A亲和层析,获得了纯度达75％以上,表达量为1 μg/mL细胞培养上清的重组融合蛋白,MTT结果显示该重组融合蛋白sPDGFRβ/Fc具有抑制PDGF刺激的Balb/c 3T3细胞增殖的能力。【结论】具有生物活性的重组可溶性受体融合蛋白sPDGFRβ/Fc可在昆虫细胞中成功地得到表达。     

莱克多巴胺核酸适配体电化学生物传感器的研制

莱克多巴胺(RAC)被大量非法用于畜牧生产,易在动物组织残留,对人体造成危害。因此,研发灵敏、快捷的检测RAC的新方法是有效控制RAC滥用的关键环节之一。通过等温滴定量热法筛选到了一条对莱克多巴胺有高亲和力(Kd=1.66×10-6mol/L)的核酸适配体,利用该适配体作为识别分子成功的构建了莱克多巴胺适配体电化学生物传感器。差分脉冲伏安法分析,在0.5~1.0×102ng/ml浓度范围内,峰电流值的差值ΔIp与莱克多巴胺浓度的对数呈现良好的线性关系,相关系数R2=0.977 0,检测限达到0.1 ng/ml,反应时间为15 min。对同一浓度的莱克多巴胺重复检测7次,其峰电流值的RSD值为3.8%;说明该传感电极具有良好的检测重现性。不仅如此,该适配体传感器还具有良好的选择性。     

Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors

There is now accumulating evidence that bone marrow-derived mesenchymal stem cells (MSCs) make an important contribution to postnatal vasculogenesis, especially during tissue ischaemia and tumour vascularization. Identifying mechanisms which regulate the role of MSCs in vasculogenesis is a key therapeutic objective, since while increased neovascularization can be advantageous during tissue ischaemia, it is deleterious during tumourigenesis. The potent angiogenic stimulant vascular endothelial growth factor (VEGF) is known to regulate MSC mobilization and recruitment to sites of neovascularization, as well as directing the differentiation of MSCs to a vascular cell fate. Despite the fact that MSCs did not express VEGF receptors, we have recently identified that VEGF-A can stimulate platelet-derived growth factor (PDGF) receptors, which regulates MSC migration and proliferation. This review focuses on the role of PDGF receptors in regulating the vascular cell fate of MSCs, with emphasis on the function of the novel VEGF-A/PDGF receptor signalling mechanism.     

Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth

We cultured smooth muscle cells as explants from rat mesenteric arterioles (40–200m in diameter) obtained by injecting a suspension of iron oxide intraarterially and magnetically separating the arterioles after collagenase digestion of adventitial tissue. In third-passaged cells we ascertained smooth muscle purity of >98% by characteristic morphology, contraction responses, and specific immunofluorescence staining. Treatment of growth-arrested (in 0.4% fetal calf serum) cells with platelet-derived growth factor (0.3–7.5 nM) or angiotensin II (0.001–1000 nM) induced ³H-thymidine incorporation and cell proliferation in a dose-dependent manner (P<0.01). S-nitroso-N acetylpencillamine (0.05–0.5 mM), a nitric oxide-generating compound, inhibited 10% fetal calf serum-induced ³H-thymidine incorporation (P<0.05) and cell proliferation (P<0.01). The antimitogenic effect of S-nitroso-N-acetylpencillamine was significantly reduced by hemoglobin and potentiated by superoxide dismutase (P<0.01). In addition to a new technique for culturing mesenteric arteriolar smooth muscle cells, these findings provide evidence that platelet-derived growth factor, angiotensin II, and nitric oxide may be involved in their growth control.     

Nitric oxide and superoxide inhibit platelet-derived growth factor receptor phosphotyrosine phosphatases

Platelet derived growth factor receptor (PDGFR) became tyrosine autophosphorylated in rat mesangial cells shortly after platelet derived growth factor (PDGF) ligation in a tyrosine kinase inhibitor (tyrphostin AG 1296) sensitive manner. Ligand-independent, massive tyrosine PDGFR phosphorylation was achieved by diverse NO releasing compounds. Phosphorylation was slow compared to PDGF, revealed a concentration- and time-dependency, and was not mimicked by lipophilic cyclic-GMP analogues. Interleukin-1 beta/cAMP activated mesangial cells released NO and in turn showed PDGFR phosphorylation. A NO-synthase involvement was assured by L-NG-nitroarginine methyl ester inhibition. PDGFR phosphorylation was also achieved by the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. NO- and O2(.-)-evoked PGDFR phosphorylation was N-acetylcysteine reversible. Cell free dephosphorylation assays revealed PDGFR dephosphorylation by tyrosine phosphatases. Receptor dephosphorylation by cytosolic phosphatases was completed within 30 min and was sensitive to the readdition of NO donors or orthovanadate. In addition, phosphatase activity determined in a direct dephosphorylation assay using the substrate para-nitrophenyl phosphate was attenuated by NO or vanadate. We conclude that cytosolic protein tyrosine phosphatases are targeted by exogenously supplied or endogenously generated NO in mesangial cells. Radical (NO. or O2.-) formation shifts the phosphorylation--dephosphorylation equilibrium towards phosphorylation, thus integrating redox-mediated responses into established signal transducing pathways.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10⁴ HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 10⁴. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 10⁴. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

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.     

Increased fluid pinocytosis,induced by action of platelet-derived growth factor on quiescent arterial smooth muscle cells,does not require increased cholesterol biosynthesis

In the current report we provide evidence that the increased rate of cholesterol biosynthesis mediated by platelet-derived growth factor in the cell cycle of monkey (Macaca nemestrina) arterial smooth muscle cells can be separated from the increased rate of fluid pinocytosis using inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.     

Suramin binds to platelet-derived growth factor and inhibits its biological activity

The polyanion suramin was recently found to inhibit binding of 125I-PDGF (platelet-derived growth factor) to Balb/c 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited 125I-PDGF binding with a half maximum inhibition concentration of approximately 60 microM or 90 micrograms/ml in a simultaneous competition assay, it was inactive in a sequential radioreceptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I-PDGF in a dose-dependent manner, with a half maximum effective concentration of approximately 50 microM. Furthermore, suramin efficiently dissociated 125I-PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can be envisaged to induce a conformational change in the PDGF-receptor complex, resulting in an increased off-rate of the complex. Reduced 125I-PDGF binding in the presence of suramin correlated directly with a suramin dose-dependent inhibition of PDGF-induced incorporation of 3H-thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)