9 results on '"Fok K"'
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2. Immunoaffinity purification and characterization of lipoprotein-associated coagulation inhibitors from Hep G2 hepatoma, Chang liver, and SK hepatoma cells. A comparative study.
- Author
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Wun, T C, primary, Huang, M D, additional, Kretzmer, K K, additional, Palmier, M O, additional, Day, K C, additional, Bulock, J W, additional, Fok, K F, additional, and Broze, G J, additional
- Published
- 1990
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3. Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.
- Author
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Chan, S D, Antoniucci, D M, Fok, K S, Alajoki, M L, Harkins, R N, Thompson, S A, and Wada, H G
- Abstract
HER2, the erbB-2/neu proto-oncogene product, is a 185-kDa transmembrane glycoprotein related to the epidermal growth factor receptor. Overexpression of HER2 was reported in several human adenocarcinomas, including mammary and ovarian carcinomas. A family of glycoproteins, the heregulin/neu differentiation factors, was characterized and implicated as the ligands for HER2. Recently, it has been shown that HER2 alone is not sufficient to reconstitute high affinity heregulin receptors and that HER3 or HER4 may be the required components of the heregulin receptors on mammary carcinoma cells (Sliwkowski, M.X., Schaefer, G., Akita, R.W., Lofgren, J.A., Fitzpatrick, V.D., Nuijens, A., Fendly, B.M., Cerione, R.A., Vandlen, R.L., and Carraway, K.L., III (1994) J. Biol. Chem. 269, 14661-14665; Plowman, G.D., Green, J.M., Culouscou, J.-M., Carlton, G.W., Rothwell, V.M., and Buckley, W. (1993) Nature 366, 473-475). Using the Cytosensor to measure the extracellular acidification rate, we have examined the effects of recombinant human heregulin-alpha on three mammary carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7), an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1 and 293-EBNA cells stably transfected with HER2. By reverse transcription polymerase chain reaction and Western blotting, we found that the breast cells also express HER3 and that the ovarian line co-expresses the HER4 message. A dramatic increase in the acidification rate was observed for the mammary carcinoma cells co-expressing high levels of HER2 and HER3. In contrast, the ovarian cells expressing high levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells expressing HER2 alone were not responsive to heregulin. When these same transfected cells were exposed to monoclonal anti-HER2 antibody followed by anti-IgG to cause aggregation of the HER2 molecules, an increase in the acidification rate was observed, indicating coupling of transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced acidification responses. These data, together with the previously reported high affinity heregulin binding and activation of tyrosine phosphorylation in HER2 and HER3 co-transfected cells support the role of HER2 and HER3 as components of the heregulin receptor in breast cells.
- Published
- 1995
4. Sequence analysis of the COOH terminus of the alpha-chain of the fourth component of human complement. Identification of the site of its extracellular cleavage.
- Author
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Hortin, G, Chan, A C, Fok, K F, Strauss, A W, and Atkinson, J P
- Abstract
The predominant form of the fourth component of complement in human and murine plasma (C4p) has an Mr approximately 5,000 less than C4 secreted by hepatocytes or macrophages (C4s). Here we demonstrate that the difference in Mr results from excision of a peptide from the COOH terminus of the alpha-chain of C4s. The site of truncation of the alpha-chain of C4 in plasma was established by sequence analysis of the COOH-terminal cyanogen bromide fragment isolated by high performance liquid chromatography. Sequential Edman degradation, digestion with carboxypeptidase Y, analysis of amino acid composition, and partial sequence analysis of an overlapping tryptic peptide established the sequence of this peptide to be Glu-Ala-Asn-Glu-Asp-Tyr-Glu-Asp-Tyr-Glu-Tyr-Asp-Glu-Leu-Pro-Ala. Comparison of our results with reported sequences establishes the COOH-terminal alanine to be residue 748 in the alpha-chain. This identifies the site at which C4s is cleaved by an extracellular protease and suggests that the protease has an elastase-like activity.
- Published
- 1986
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5. Conformational analysis of COOH-terminal segments of human C3a. Evidence of ordered conformation in an active 21-residue peptide.
- Author
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Lu, Z X, Fok, K F, Erickson, B W, and Hugli, T E
- Abstract
Human C3a, a 77-residue fragment released during activation of the serum complement system, is a potent spasmogen that contracts a variety of smooth muscle tissues and enhances vascular permeability. Previous studies have suggested that a 5-residue, COOH-terminal segment of C3a constitutes the essential active site of this molecule; however, the pentapeptide is 1000-fold less active than C3a. Human C3a 57-77, a synthetic segment containing the 21 COOH-terminal residues of C3a, exhibits potencies nearly equivalent to those of natural C3a in several biologic assay systems. The circular dichroism spectra of synthetic peptides corresponding to sequences 57-77, 65-77, and 73-77 in human C3a were measured in water and trifluoroethanol. The CD spectra in the far-UV region indicate that each C3a peptide assumes a random coil conformation in aqueous solution with little evidence of alpha-helical structure. However, C3a peptide 57-77 assumes predominantly an alpha-helical conformation (47%) in 25% trifluoroethanol, while the shorter tridecapeptide 65-77 and pentapeptide 73-77 appear by CD to contain beta-turn conformations only Crystallographic analysis of human C3a indicated that the NH2-terminal portion of peptide 57-77 adopts an alpha-helical structure and that the COOH-terminal portion, including residues 73-77, contains an irregular fold much like a beta-turn. Since C3a peptide 57-77 exhibits activities qualitatively and quantitatively similar to natural C3a, we propose that this synthetic peptide adopts a helical conformation when bound to its cellular receptor which corresponds to that in the intact C3a molecule. Consequently, the NH2-terminal portion (residues 1-21) and the disulfide-linked core region (residues 22-57) in intact C3a serve primarily to stabilize ordered conformation in the COOH-terminal region (residues 58-77) and thereby orient side chains at the essential active site for optimal receptor interaction.
- Published
- 1984
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6. Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming growth factor beta. Immunohistochemical localization and association with differentiating cells.
- Author
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Ellingsworth, L R, Brennan, J E, Fok, K, Rosen, D M, Bentz, H, Piez, K A, and Seyedin, S M
- Abstract
Two apparently homologous proteins, designated CIF-A and CIF-B, were previously isolated from bovine bone on the basis of their cartilage-inducing activity in culture. CIF-A has been shown to probably be identical to transforming growth factor beta (TGF-beta). To address the question of tissue localization, antibodies to CIF-A were produced using a synthetic polypeptide identical to N-terminal residues 1-30. The antibodies were immunoreactive with bovine CIF-A and human TGF-beta, did not recognize CIF-B, and did not recognize other molecular weight species in crude bovine bone extracts. The antibodies were used to immunohistochemically localize CIF-A/TGF-beta in fetal bovine bone and other tissues. There was abundant staining of osteocytes throughout cancellous and cortical bone as well as chondrocytes within the articular cartilage, although growth plate-associated chondrocytes were not labeled. In addition, immunoreactive cells were detected in bone marrow (megakaryocytes and some mononuclear cells), fetal liver (hematopoietic stems cells), and the thymus (Hassall's corpuscle and some medullary thymocytes). In the kidney, the antibodies labeled a population of epithelial cells lining the calyces. Tissues which did not have detectable amounts of CIF-A/TGF-beta included the thyroid, adrenal, salivary gland, and aorta. Results presented here suggest that the factor may function in vivo as a general development and repair factor and may play a significant role in the differentiation of many cell types including chondrocytes, osteocytes, T-lymphocytes, and red blood cells.
- Published
- 1986
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7. Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate.
- Author
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Hills R, Mazzarella R, Fok K, Liu M, Nemirovskiy O, Leone J, Zack MD, Arner EC, Viswanathan M, Abujoub A, Muruganandam A, Sexton DJ, Bassill GJ, Sato AK, Malfait AM, and Tortorella MD
- Subjects
- ADAM Proteins genetics, ADAMTS4 Protein, Amino Acid Motifs, Amino Acid Sequence, Extracellular Matrix Proteins genetics, Glutamic Acid metabolism, Inhibitory Concentration 50, Kinetics, Matrilin Proteins, Molecular Sequence Data, Peptides chemical synthesis, Peptides chemistry, Procollagen N-Endopeptidase genetics, Protein Array Analysis, Protein Isoforms chemistry, Protein Isoforms metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Spectrometry, Fluorescence, Substrate Specificity, ADAM Proteins metabolism, Extracellular Matrix Proteins metabolism, Peptide Library, Peptides metabolism, Procollagen N-Endopeptidase metabolism
- Abstract
ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.
- Published
- 2007
- Full Text
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8. Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes.
- Author
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Tortorella MD, Arner EC, Hills R, Easton A, Korte-Sarfaty J, Fok K, Wittwer AJ, Liu RQ, and Malfait AM
- Subjects
- ADAM Proteins, ADAMTS4 Protein, ADAMTS5 Protein, Amino Acid Sequence, Animals, Binding Sites, Blotting, Western, Cattle, Cell Line, Dose-Response Relationship, Drug, Drosophila, Epitopes chemistry, Glutamic Acid chemistry, Humans, Kinetics, Metalloendopeptidases antagonists & inhibitors, Methylamines chemistry, Molecular Sequence Data, Nasal Septum metabolism, Peptides chemistry, Procollagen N-Endopeptidase antagonists & inhibitors, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Synovial Fluid metabolism, Time Factors, Tissue Inhibitor of Metalloproteinase-3 metabolism, alpha-Macroglobulins metabolism, Enzyme Inhibitors chemistry, Metalloendopeptidases metabolism, Procollagen N-Endopeptidase metabolism, alpha-Macroglobulins physiology
- Abstract
Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.
- Published
- 2004
- Full Text
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9. Sulfation of a tyrosine residue in the plasmin-binding domain of alpha 2-antiplasmin.
- Author
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Hortin G, Fok KF, Toren PC, and Strauss AW
- Subjects
- Amino Acid Sequence, Amino Acids metabolism, Binding Sites, Chromatography, High Pressure Liquid, Cyanogen Bromide, Humans, Hydrolysis, Peptide Fragments metabolism, Trypsin, Fibrinolysin metabolism, Sulfates metabolism, Tyrosine metabolism, alpha-2-Antiplasmin metabolism
- Abstract
Sulfation of human alpha 2-antiplasmin, the major plasma inhibitor of fibrinolysis, was examined using both protein isolated from human plasma and protein synthesized and biosynthetically labeled with [35S]sulfate by a human hepatoma-derived cell line. Linkage of sulfate to tyrosine was demonstrated by recovery of labeled tyrosine sulfate after base hydrolysis of sulfate-labeled alpha 2-antiplasmin. Analysis by reverse-phase high performance liquid chromatography of peptides released from alpha 2-antiplasmin by cleavage with trypsin or cyanogen bromide indicated that sulfate is linked to a single segment of the protein. A cyanogen bromide peptide corresponding to the sulfate-labeled peptide was prepared from alpha 2-antiplasmin isolated from human plasma. Consistent with the presence of tyrosine sulfate in this peptide, its chromatographic elution was altered by treatment with acid under conditions which release sulfate from a tyrosine residue. No peptide in the total digest of alpha 2-antiplasmin by cyanogen bromide eluted at the position of the peptide following desulfation, suggesting that all of the protein is in a sulfated form. The sequence of the sulfate-containing cyanogen bromide peptide as determined by sequential Edman degradation, amino acid composition, and fast atom-bombardment-mass spectrometry was: Glu-Glu-Asp-Tyr(SO4)-Pro-Gln-Phe-Gly-Ser-Pro-Lys-COOH. This peptide is a segment of the previously identified plasmin-binding domain of alpha 2-antiplasmin.
- Published
- 1987
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