Biomedical subjects

M D Kuo

Publications and source records attributed to M D Kuo.

At least 19 recordsLinked to original sources

PubMed2001

Role of reactive oxygen intermediates in Japanese encephalitis virus infection in murine neuroblastoma cells.

Infection with Japanese encephalitis virus (JEV), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. This study attempted to determine whether JEV infection induces free radical generation and whether oxidative stress contributes to virus-induced cell death in neuroblastoma cells. A rise in the intracellular level of free radicals indicated by the 2',7'-dichlorofluorescein fluorescence was observed in N18 cells following JEV infection. Cellular flavon-containing enzymes were involved in JEV-induced fluorescent change. Cells were moderately protected from JEV-induced death by diphenyleneiodonium, a flavon-containing enzyme inhibitor, whereas common antioxidants such as N-acetylcysteine, pyrrolidine dithiocarbamate, Tiron, and Trolox turned out to be ineffective. These results suggest that the direct antioxidant action is not helpful in prevention of JEV-induced neuronal cell death.

Animals↗

PubMed2001

Therapeutic elastase inhibition by alpha-1-antitrypsin gene transfer limits neointima formation in normal rabbits.

PURPOSE: Alpha-1-antitrypsin (AAT) is the major circulating elastase inhibitor. Deficiency of elastase inhibition leads to emphysema and vascular abnormalities including accelerated neointima. Because recent evidence suggests that tissue AAT levels determine inhibitory function, the authors hypothesize that local tissue-based expression of AAT limits elastase activity sufficiently to guide arterial response to injury. MATERIALS AND METHODS: Rabbit common femoral arteries were injured by mechanical overdilation and treated with buffer, viral control, or an adenovirus expressing AAT (Ad/AAT). After 3 and 28 days, intima-to-media (I/M) ratios were evaluated. Additionally, early changes in elastase inhibition potential (3 d), extracellular elastin and collagen content (3 d), and local macrophage and neutrophil infiltration (7 d) were determined. RESULTS: Ad/AAT significantly decreased neointima formation after mechanical dilation injury after 28 days: buffer controls exhibited mean I/M ratios of 0.76 +/- 0.06, whereas viral controls reached 0.77 +/- 0.09; in contrast, Ad/AAT reduced I/M ratios to 0.44 +/- 0.06. Both early elastin and collagen content were preserved in the Ad/AAT group relative to controls. The Ad/AAT group also reversed the local inflammation that characterized viral controls. CONCLUSIONS: This strategy demonstrates that local increases in elastase inhibition potential promote a neointima-resistant small-caliber artery, which may offer new promise in management of patients undergoing angioplasty.

Angioplasty↗

PubMed2001

Enhancement of neointima formation with tissue-type plasminogen activator.

PURPOSE: Indirect evidence suggests that tissue plasminogen activator (tPA) either limits or does not alter restenosis. However, tPA enhances tumor invasiveness through matrix remodeling, and several elements of degraded matrix enhance smooth muscle cell mitogenesis. We use either local adenoviral-mediated overexpression of tPA or systemic infusion of recombinant tPA combined with mechanical overdilation of rabbit common femoral arteries to evaluate the impact of tPA on neointima formation. METHODS: Left common femoral arteries of New Zealand white rabbits were transfected in situ either with an adenoviral-construct-expressing tPA or a viral control (adenoviral-construct-expressing beta-galactosidase) or nonviral (buffer) control after balloon angioplasty injury. At 7 and 28 days, left common femoral artery segments were harvested (n = 4 for each group and time point). Vessel segments were examined for intimato-media ratio, smooth muscle cell proliferation, extracellular matrix, and inflammatory response. Thrombus formation was evaluated after 3 days (n = 3 for each group). In a second experiment, New Zealand white rabbits (n = 3 per group, per time point) underwent mechanical dilation followed by buffer treatment or systemic tPA infusion according to a widely clinically used accelerated infusion protocol. Treated artery segments were harvested after 7 or 28 days and processed for intima-to-media ratio determination and class-wide histochemical determination of collagenous extracellular matrix and collagen content. RESULTS: Both rate and degree of neointima formation increase dramatically with overexpression (250%-461% relative to controls at 7 and 28 days). Substantial early matrix degradation is observed in vessels treated with local overexpression of tPA, although no increases in local inflammation or in smooth muscle proliferation occur. Late enhancement of smooth muscle proliferation emerges, consistent with secondary impact of perturbed matrix components. Systemic infusion of tPA according to clinical protocols also results in early and late enhancement of neointima formation in this model (34%-52% relative to controls at at 7 and 28 days), with significant early collagenous matrix degradation. Systemic infusion, although significant, did not attain the degree of neointima formation present with overexpression. CONCLUSION: With some evidence of dose-dependence, tissue plasminogen activator enhances neointima formation after angioplasty in a rabbit model. Early matrix degradation precedes change in rates of proliferation and underlies this effect in spite of several antirestenotic actions including decreased thrombus and decreased macrophage recruitment in this model.

Adenoviridae↗

PubMed2001

High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen.

The prevalence of NS1 Ab response in patients with dengue viral infection and the potential of using recombinant NS1 protein as a diagnostic antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal half of NS1 proteins (rNS1, rNS1-C) were highly expressed (10-30 mg/l) and further purified and refolded. The good antigenicity of the full-length rNS1 protein was confirmed by interaction with 19 dengue NS1-specific monoclonal antibodies (MAbs) in ELISA; however, the antigenicity of rNS1-C was relatively lower. The full-length rNS1 antigen also differentiated reliably between sera from dengue virus-infected patients and sera from normal controls. When rNS1 was used as an antigen to detect human anti-NS1 IgM and IgG Ab, the anti-NS1 Ab response was found in 15 of 17 patients (88%) with primary dengue infection and all 16 patients (100%) with secondary dengue infection. These results indicated that using the full-length rNS1 whose antigenicity is restored as ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either primary or secondary dengue infection. This finding suggested that the anti-NS1 antibody appeared not only in secondary and severe dengue virus infection and might not correlate the pathogenesis of dengue hemorrhagic fever. The study also verified that our purified rNS1 protein showed similar immunological properties as native dengue viral proteins. Genetic engineering production of recombinant NS1 antigen could provide a safe and valuable resource for dengue virus serodiagnosis.

Animals↗

PubMed2000

Thrombomodulin overexpression to limit neointima formation.

BACKGROUND-These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury. METHODS AND RESULTS-An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76+/-0.06%, whereas viral controls reached 0.77+/-0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47+/-0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals. CONCLUSIONS-This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.

Animals↗

PubMed2000

Astrocytic alteration induced by Japanese encephalitis virus infection.

The neurotropism of Japanese encephalitis virus (EV) has not been well characterized. Astrocytes are parts of the blood-brain barrier, a major source of chemokines, and critical effectors of central inflammation. Thus, astrocytes might play some role as JEV travels from the peripheral to the CNS and/or the resultant encephalitis. Using rat cortical cultures, we found that JEV can cause cellular and/or functional changes in astrocytes as indicated by increased expression of interleukin-6 (IL-6), regulated by activation, normal T cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1), increased lactate release and glucose uptake, and attenuation of glutamate toxicity. These modulations occur needed by the cells for compensation and may affect neuron and/or astrocyte function.

Animals↗

PubMed1999

Gene therapy to promote thromboresistance: local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model.

Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis. To examine the impact of local tPA overexpression, an adenoviral vector expressing tPA was created. The construct was characterized functionally in vitro, and the function of the vector was confirmed in vivo by delivery to the rabbit common femoral artery. Systemic coagulation parameters were not perturbed at any of the doses examined. The impact of local overexpression of tPA on in vivo thrombus formation was examined subsequently in a stasis/injury model of arterial thrombosis. The construct effectively prevented arterial thrombosis in treated animals, whereas viral and nonviral controls typically developed occluding thrombi. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery.

Adenoviridae↗

PubMed1998

1998 ARRS President's Award. The potential of in vivo vascular tissue engineering for the treatment of vascular thrombosis: a preliminary report. American Roentgen Ray Society.

OBJECTIVE: Current gene therapy and tissue engineering protocols suffer from a number of inherent limitations. In this study, we examine the feasibility of a new approach for the treatment of vascular thrombosis: in vivo tissue engineering. MATERIALS AND METHODS: Rabbit femoral veins were transfected in situ with either a previously characterized adenoviral-construct-expressing tissue plasminogen activator or a viral (adenoviral-construct-expressing beta-galactosidase) or nonviral (buffer) control and used as cross sections (n = 3). Treated veins were then harvested and grafted into the ipsilateral common femoral artery as an interposition vein graft. A potent stimulus for thrombus formation was then introduced into the recipient artery downstream of the graft. Six days later, the rabbits were sacrificed, and the grafts and downstream arteries were harvested. Vessel segments were then examined for thrombus according to defined anatomic zones. Transfection efficiency and presence of smooth muscle cells in the vein graft were also evaluated. RESULTS: The engineered vein graft showed a significant reduction in thrombus formation within both the graft and the downstream artery relative to nonviral (buffer) and viral (adenoviral-Rous sarcoma virus beta-galactosidase [Adv/RSV-betagal]) controls. Underlying endothelial cell transfection efficiency of 90% was observed in viral controls (Adv/RSV-betagal). A 2.4-fold increase in smooth muscle alpha-actin positive cells in the engineered vein graft was seen compared with nonviral (phosphate-buffered saline) controls. A 10-fold increase in smooth muscle alpha-actin-positive cells in the engineered vein graft relative to viral (Adv/RSV-betagal) controls was also observed. CONCLUSION: In vivo tissue engineering is a new paradigm in molecular medicine that is a viable alternative to conventional gene therapy and tissue engineering for the treatment of vascular thrombosis.

Animals↗

PubMed1998

In vivo CT and MR appearance of prosthetic intraocular lens.

PURPOSE: We present the first in vivo CT and MR imaging description of intraocular lenses (IOLs), which are commonly encountered in elderly patients who have undergone cataract surgery. METHODS: A retrospective review was done of the imaging studies of 20 patients (22 eyes) with IOLs and of three patients (four eyes) with aphakia. CT and MR studies were performed with standard clinical protocols. RESULTS: Sixteen patients with 18 posterior IOLs underwent six CT and 43 MR studies. Four patients with four anterior IOLs had one CT and eight MR studies. The exact position of the optic portion of the IOL could be optimally determined on CT scans with 1-mm-thick sections and on fat-saturated fast T2-weighted MR orbital coil studies performed on a 1.5-T imager. The haptics could not be distinguished from the ciliary body. Three patients with aphakia had eight MR and two CT studies. Aphakia was difficult to identify if the image thickness was greater than the diameter of the pupil (2.5 to 4.0 mm). CONCLUSION: The optic portion of an IOL is visible on either high-quality CT or MR studies. However, the haptic portion is not visible on clinical in vivo images.

Aphakia↗

PubMed1997

RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.

The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3' end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3' NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.

Binding Sites↗

PubMed1996

Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein.

Japanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (delta 1-148 and delta 1-323) forms of NS3 were engineered and expressed in E. coli as fusion proteins with a histidine tag at the N terminus. The purified recombinant proteins his-NS3 and his-NS3(delta 1-148) were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3(delta 1-323) did not. The requirements for MgCl2 and MnCl2 and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses. Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases. The substrate preference was in the order GTP > ATP >> UTP > CTP. Interestingly, we found that Ca2+ could not substitute for Mg2+; on the contrary, it inhibited NTPase activity. The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148-323) completely abolished the activity. Therefore, amino acids 148-323 contain a critical region required for NTPase activity.

Acid Anhydride Hydrolases↗

PubMed1995

Identification, purification, and characterization of cell-surface retention sequence-binding proteins from human SK-Hep cells and bovine liver plasma membranes.

Cell-surface retention is a newly identified mechanism associated with the secretion of certain polypeptide growth factors and cytokines. This novel form of secretion appears to be mediated by cell-surface retention sequences (CRS) in the polypeptide molecules. To test the hypothesis that high-affinity CRS-binding proteins (CRS-BPs) are responsible for the cell-surface retention, we identified and characterized the high-affinity binding sites on various cell types for 125I-labeled CRS peptide (sis) and CRS peptide (VEGF), each of which contained the putative CRS motifs of platelet-derived growth factor B (c-sis) and vascular endothelial cell growth factor, respectively. Scatchard plot analysis revealed a single class of high-affinity binding sites with Kd = 0.5-0.7 nM and approximately 22,000-55,000 sites/cell. High-affinity binding activity could be demonstrated between pH 4.5 and 8.0, but was much greater below 6.0 (maximum pH 5.0-5.5). The ligand binding activity was inhibited by heparin, polylysine, and protamine, but not by cytochrome c. CRS-BPs responsible for the high-affinity binding were identified as 60-72-kDa proteins by ligand affinity labeling. CRS-BPs were purified from human SK-Hep cells and bovine liver plasma membranes by Triton X-100 extraction followed by affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. Purified CRS-BPs exhibited ligand binding properties (pH profile and inhibitor sensitivity) similar to those of the high-affinity binding sites for CRS peptides on cultured cells. The major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have identical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS-BP1.

Amino Acid Sequence↗

PubMed1993

The major acidic fibroblast growth factor (aFGF)-stimulated phosphoprotein from bovine liver plasma membranes has aFGF-stimulated kinase, autoadenylylation, and alkaline nucleotide phosphodiesterase activities.

The major acidic fibroblast growth factor (aFGF)-stimulated phosphoprotein (MAFP) purified from bovine liver exhibits kinase, autoadenylylation, and alkaline nucleotide phosphodiesterase activities depending upon reaction conditions. In the presence of divalent ions, MAFP showed intrinsic and a FGF-stimulated kinase activities (autophosphorylation) using either [gamma-32P]ATP or [gamma-32P]GTP as a substrate. The autophosphorylation activity of MAFP was stimulated at low concentrations of Ca2+, Mg2+, or Mn2+ (0.2-2 microM). Depletion of the divalent ions by EDTA abolished the autophosphorylation activity but enhanced the autoadenylylation activity of MAFP. [alpha-32P]ATP as well as [alpha-32P]NAD could serve as substrates for autoadenylylation activity of MAFP. aFGF appeared to enhance the autoadenylylation activity of MAFP with an optimal concentration (0.6-1.2 nM). P1, P3-di(adenosine-5')-triphosphate (AP3A) was found to be a potent inhibitor for the autophosphorylation and autoadenylylation activities of MAFP. Analyses by automated Edman degradation of the adenylylated and phosphorylated peptides derived from autoadenylylated and autophosphorylated MAFP revealed that both autoadenylylation and autophosphorylation occurred at residue Thr204. The kinase and autoadenylylation activities of MAFP had an optimal pH of 6.9-7.4. However, at pH 8.9, MAFP showed intrinsic and aFGF-stimulated phosphodiesterase activities. aFGF appeared to stimulate the phosphodiesterase activity of MAFP without altering the Km (approximately 0.2 mM) of its substrate.

Adenine↗

PubMed1992

Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells.

Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types.

3T3 Cells↗

PubMed1991

The plasma cell membrane glycoprotein, PC-1, is a threonine-specific protein kinase stimulated by acidic fibroblast growth factor.

A 32P-labeled protein that co-purified with acidic fibroblast growth factor (aFGF) receptor from bovine liver proved to be a distinct membrane protein, which itself has kinase activity that is stimulated by aFGF. The protein was designated MAFP for major aFGF-stimulated phosphoprotein. MAFP was purified from bovine liver using immunoaffinity chromatography with monoclonal antibody to MAFP following Triton X-100 extraction of plasma membranes and wheat germ lectin-Sepharose 4B column chromatography. The purified MAFP showed molecular masses of 130 kDa and 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. Purified MAFP elicited aFGF-stimulated Thr-specific autophosphorylation activity and phosphorylation activity toward protein substrates (myelin basic protein and histone). Amino acid sequence analyses of 16 peptide fragments of MAFP, produced by endoproteinase Lys-C digestion followed by reduction and S-pyridylethylation, showed approximately 80-100% homology with the cDNA-deduced amino acid sequences of human and mouse plasma cell membrane glycoprotein, PC-1 (Buckley, M. F., Loveland, K. A., McKinstry, W. J., Garson, O. M., and Goding, J. W. (1990) J. Biol. Chem. 265, 17506-17511), suggesting that MAFP is the bovine version of PC-1. The amino acid sequences of bovine MAFP, human and mouse PC-1 reveal a putative ATP binding site in their extracellular domains. These results suggest that MAFP(PC-1) is an ectoprotein kinase. In addition to the kinase activity, MAFP(PC-1) was also found to possess alkaline nucleotide phosphodiesterase activity. It is now clear that several of the unique properties previously attributed to the aFGF receptor kinase are actually properties of this novel Thr-specific ectoprotein kinase, which co-purifies with the aFGF receptor and is responsive to stimulation by aFGF.

Amino Acid Sequence↗

PubMed1991

Purification of a new type high molecular weight receptor (type V receptor) of transforming growth factor beta (TGF-beta) from bovine liver. Identification of the type V TGF-beta receptor in cultured cells.

A 400-kDa transforming growth factor beta (TGF-beta) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B affinity chromatography, DEAE-cellulose anion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. This procedure yielded approximately 20 micrograms of the receptor from 1 kg of bovine liver. During purification, the 400-kDa TGF-beta receptor was detected by a cross-linking assay in which the TGF-beta receptor-125I-TGF-beta complex was cross-linked by disuccinimidyl suberate, a bifunctional reagent, and analyzed by 5.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. This novel 400-kDa TGF-beta receptor was also identified on cultured cells including cells reported to lack the type III receptor. The 400-kDa TGF-beta receptor, a nonproteoglycan glycoprotein, appears to be distinct from TGF-beta receptors (types I, II, III, and IV) previously identified on cultured cells and is designated as the type V receptor. The 400-kDa TGF-beta receptor as well as type I, II, and III receptors underwent internalization upon 125I-TGF-beta binding in mink lung epithelial cells.

Amino Acid Sequence↗

PubMed1990

Amino acid sequence and characterization of a heparin-binding neurite-promoting factor (p18) from bovine brain.

A neurite-promoting factor (p18) was isolated from bovine brain using ammonium sulfate fractionation, sulfated Sephadex G-50 chromatography, heparin-Sepharose gel chromatography, and reverse phase high performance liquid chromatography. The complete amino acid sequence of p18 was determined by automated Edman degradation of S-pyridylethylated p18, and its peptide fragments produced by cyanogen bromide cleavage and by digestion with specific endoproteinases. Alignment of the amino acid sequences of these peptides revealed that p18 consists of 119 amino acid residues with a calculated molecular weight of 14,200. p18 appears to possess five disulfide bonds per molecule. A region of amino acid sequence at the C terminus of p18 shows a structural feature homologous to that around the reactive sites of Kazal-type protease inhibitors. However, p18 did not exhibit anti-trypsin activity. p18 showed very little, if any, mitogenic activity toward NIH 3T3 cells and Swiss mouse 3T3 cells. p18 was found to bind to a specific receptor with an apparent Kd of approximately 8 nM and receptor numbers 1.7 x 10(5) and 1.0 x 10(4) for NIH 3T3 cells and PC12 cells (rat pheochromocytoma cells), respectively.

Amino Acid Sequence↗

PubMed1990

Acidic fibroblast growth factor receptor purified from bovine liver is a novel protein tyrosine kinase.

Acidic fibroblast growth factor (aFGF) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B gel affinity chromatography, and DEAE-cellulose anion-exchange chromatography. As previously reported for the aFGF receptor in murine fibroblasts (Huang, S. S., and Huang, J. S. (1986) J. Biol. Chem. 261, 9568-9571), the purified aFGF receptor was also found to be a 135-kDa glycoprotein which showed an intrinsic and ligand-stimulated autophosphorylation activity. The 32P-labeled aFGF receptor was specifically immunoprecipitated by anti-FGF receptor (anti-flg/bek/cek gene product) antiserum. In contrast to other growth factor receptors/protein tyrosine kinases, the protein tyrosine kinase activity (autophosphorylation) of the aFGF receptor was stimulated (approximately 1.5-fold) by low concentrations of Mn2+, Mg2+, and Ca2+ (optimal concentrations of approximately 0.1, approximately 0.1, and 1 microM, respectively) but inhibited by higher concentrations of Mn2+, Mg2+, Ca2+, and pyrophosphate (greater than or equal to 20, greater than or equal to 50, greater than or equal to 10, and greater than or equal to 100 microM, respectively). However, addition of Mn2+ and pyrophosphate at a ratio of 1:1 not only reversed the inhibitory effect but also enhanced the kinase activity about 3-4-fold. The apparent Km of ATP for intrinsic and ligand-stimulated protein kinase activity of the aFGF receptor was estimated to be 25 microM. The preferred exogenous substrates for the protein tyrosine kinase activity of the aFGF receptor were found to be myelin basic protein and histone. Poly-L-arginine, an inhibitor for aFGF binding to the receptor, appeared to stimulate the mitogenesis or cell growth of responsive cells by mimicking aFGF activity.

Amino Acid Sequence↗