Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent Mr = 36,300 under reducing conditions on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly-Pro-Pro-Pro- Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly-Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.
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PMID:Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells. 242 67

Previously, monoclonal antibody FDC-6 was established, which defines a structure specific for fibronectins isolated from fetal and malignant cells and tissues. The presence of the FDC-6-defined structure at type III connecting segment (III CS) is characteristic of oncofetal fibronectin (onf-FN), and its absence is characteristic of normal fibronectin (nor-FN) (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). Hepatoma fibronectin was sequentially digested by various proteases, followed by subsequent chromatography on an FDC-6 affinity column and reverse-phase columns at each step of digestion. A single strongly active glycosylhexapeptide (glycopeptide 1) and an inactive glycosylpentapeptide (glycopeptide 3) were isolated from glycopeptide A containing 35 amino acid residues. The minimum essential structure required for the FDC-6 activity was found to be a hexapeptide sequence Val-Thr-His-Pro-Gly-Tyr having NeuAc alpha 2----3Gal beta 1----3GalNAc or its core (Gal beta 1----3GalNAc or GalNAc) linked at threonine. Various synthetic peptides including the Val-Thr-His-Pro-Gly-Tyr sequence and a glycopeptide having the Val-Thr-His-Pro-Gly pentapeptide with the same glycosylation at threonine were all inactive. Elimination of sialic acid slightly increased the activity, and subsequent elimination of galactose did not alter the activity; however, removal of the Gal beta 1----3GalNAc residue by endo-alpha-N-acetylgalactosaminidase from desialylated glycopeptide A resulted in total inactivation of the reactivity with FDC-6 antibody. Thus, a single glycosylation at a defined threonine residue of the III CS region may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. This finding opens the possibility that a number of other oncofetal epitopes consist of a peptide and a common O-linked carbohydrate and that the combination produces a conformation specific to cancer or to a stage of development.
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PMID:The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6. Unique structural requirement for the antigenic specificity provided by a glycosylhexapeptide. 244 38

The low-molecular-mass insulin-like growth factor-binding protein (IGF-BP) and placental protein 12 (PP12) are identical proteins that are present in human serum, amniotic fluid, secretory endometrium and decidua. IGF-BP/PP12 is believed to act as an autocrine or paracrine regulator of cell growth. A cDNA clone encompassing the entire protein coding region of this protein was isolated from a human decidual cDNA library. The authenticity of the cDNA was verified by in vitro transcription/translation experiments and by the identity of the 10 N-terminal amino acids deduced for the mature peptide with those obtained by direct protein sequencing. The amino acid sequence indicates that pre-IGF-BP/PP12 consists of 259 amino acid residues. The putative signal peptide is 25 residues long, and the mature protein thus contains 234 amino acids and has a molecular mass of 25293 Da. The sequence is very cysteine-rich at the N-terminus after which there are regions of clustered Pro, Glu, Ser and Thr residues (so-called PEST regions), which exist in proteins with short half-lives. The amino acid sequence also includes an Arg-Gly-Asp tripeptide that may function as a cell recognition signal. The IGF-BP/PP12 gene encodes a single 1.6 kb mRNA species that is expressed in decidua, secretory endometrium, liver and a human hepatoma cell line (HepG2). Southern blot analysis suggests that there is a single IGF-BP/PP12 gene in the human genome.
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PMID:Primary structure of human insulin-like growth factor-binding protein/placental protein 12 and tissue-specific expression of its mRNA. 245 13

The monoclonal antibody FDC-6 defines a structure specific to oncofetal fibronectins (onf-FN) isolated from fetal and malignant cells and tissues. The absence of this structure is characteristic of normal fibronectin (nor-FN) isolated from plasma and adult normal tissue (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). The minimum structure required for FDC-6 reactivity was determined to be Val-Thr-His-Pro-Gly-Tyr (VTHPGY) with alpha-N-acetylgalactosamine (alpha-GalNAc) at Thr, although alpha-GalNAc per se is not involved in the FDC-6 epitope (Matsuura, H., Takio, K., Titani, K., Greene, T., Levery, S. B., Salyan, M. E. K., and Hakomori, S. (1988) J. Biol. Chem. 263, 3314-3322). Thus, a single glycosylation on the normally occurring peptide of FN may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. The FDC-6-nonreactive synthetic peptide containing the VTHPGY sequence was converted into FDC-6-reactive form on incubation with alpha-N-acetylgalactosaminyltransferase and UDP-[3H]GalNAc in the homogenate of hepatoma cell HUH-7, human fetal fibroblast cell line WI-38, or human epidermoid carcinoma cell line A431. Such a conversion did not take place when the same enzyme fraction of normal adult tissue was incubated with the VTHPGY peptide under the same conditions. Thus, the occurrence of alpha-GalNAc transferase recognizing the VTHPGY peptide sequence (UDP-GalNAc:VTHPGY alpha-GalNAc transferase) is specific for fetal and cancer tissues, and absent in normal adult tissues. However, a similar alpha-GalNAc transferase activity capable of transferring the GalNAc residue to other Ser or Thr hydroxyl groups of nor-FN, and presumably located at the type III connecting segment region, was detectable in homogenate of various normal tissues. Such enzyme activity was determined with the use of enzymatically de-O-glycosylated nor-FN. Thus, the enzymatic basis of FDC-6 epitope formation is a subtle change in the substrate specificity of alpha-GalNAc transferase. The normal enzyme is incapable of transferring alpha-GalNAc from UDP-GalNAc to the Thr residue of the VTHPGY sequence, but is capable of transferring alpha-GalNAc to other Ser or Thr residues of FN. In contrast, alpha-GalNAc transferase of fetal and cancer tissues may have broader specificity and the capability to transfer GalNAc to Thr or Ser residues, including those of the VTHPGY sequence.
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PMID:An alpha-N-acetylgalactosaminylation at the threonine residue of a defined peptide sequence creates the oncofetal peptide epitope in human fibronectin. 247 5

Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti-(P-Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half-maximal and maximal binding of the antibodies to plates coated with BSA - P-Thr occurred at serum dilutions of 1:4000 and 1:1000, respectively. P-Thr inhibited antibody binding with a half-maximal effect at 40 microM. P-Ser was 200-fold less potent while P-Tyr was essentially ineffective. Anti-(P-Thr) antibodies could specifically bind to phosphothreonine-containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P-Thr at 10 microM completely inhibited antibody binding while P-Ser, P-Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti-(P-Thr) antibodies were also capable of specifically immunoprecipitating 32P-labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 mM P-Thr but not by P-Tyr. These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.
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PMID:Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation. 250 Mar 41

In two-dimensional tryptic phosphopeptide mapping, the beta-subunit of the insulin receptor phosphorylated by 12-O-tetradecanoylphorbol-13-acetate in rat hepatoma cells (H-35) was separated into one phosphothreonine-containing peptide and several phosphoserine-containing peptides. The synthetic peptide coding residues 1327-1343 in the C-terminal region of the rat insulin receptor was phosphorylated at the threonine residue by protein kinase C in a phosphatidylserine and oleoylacetylglycerol dependent manner. Tryptic digest of this phosphopeptide migrated to the same position as the phosphothreonine containing peptide obtained from the beta-subunit in two-dimensional phosphopeptide mapping. These data suggested that Thr 1336 of the insulin receptor is the site of phosphorylation by protein kinase C in intact cells.
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PMID:Identification of a phosphorylation site of the rat insulin receptor catalyzed by protein kinase C in an intact cell. 250 75

A phosphorylated basic fibroblast growth factor (FGF) can be detected in extracts of bovine capillary endothelial cells and human hepatoma cells. Accordingly, human basic FGF contains consensus sequences that account for its phosphorylation on Thr-112 by the catalytic subunit of the cAMP-dependent protein kinase A (PK-A) and on Ser-64 by the calcium- and phospholipid-dependent protein kinase C (PK-C). A kinetic analysis of both of these reactions revealed that basic FGF is among the better substrates for these enzymes. Although the kinase responsible for the phosphorylation in vivo has not yet been identified, we examined the effects of phosphorylation on the biological activity, heparin-binding capacity, and receptor-binding capacity of phosphorylated basic FGF. No effects of phosphorylation were observed when the mitogen was phosphorylated by PK-C. In contrast, when basic FGF was phosphorylated in the receptor-binding domain with PK-A, the growth factor was 3-8 times better at displacing radiolabeled basic FGF in the radioreceptor assay. No effects were seen on the binding of this FGF to immobilized heparin or cell-associated glycosaminoglycans, suggesting that this phosphorylation modifies the affinity of basic FGF for its receptor. Biological assays for basic FGF failed to identify differences between the phosphorylated and unphosphorylated forms of recombinant basic FGFs presumably because of the presence of ectophosphatases and the experimental conditions of proliferation and mitogenic assays (37 degrees C, 24-96 hr). Because the relative affinity of basic FGF for its receptor and cell-associated glycosaminoglycans may regulate its activity, the identification of a modified form of basic FGF may be of particular importance in understanding the mechanisms that regulate its biological activity, bioavailability, and processing to and from the extracellular matrix.
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PMID:Basic fibroblast growth factor is a substrate for protein phosphorylation and is phosphorylated by capillary endothelial cells in culture. 254 33

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.
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PMID:Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling. 255 Apr 26

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.
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PMID:Isolation and sequence analysis of a cDNA clone encoding the fifth complement component. 257 66

The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases.
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PMID:Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease. 265 1


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