Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0025202 (melanoma)
 69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyrosine countertransport was used to demonstrate the existence of a carrier system for neutral amino acids in the lysosomal membrane of FRTL-5 thyroid cells. In addition to tyrosine, the carrier system recognized the neutral amino acids leucine, histidine, phenylalanine, and tryptophan. Cystine and lysine, amino acids for which a lysosomal carrier system has been demonstrated, showed no competition with tyrosine for countertransport. The tyrosine system showed stereospecificity and cation independence. It did not require an acidic lysosome or the availability of free thiols. The apparent Km for tyrosine was approximately 100 microM; the energy of activation of the system was approximately 9.7 kcal/mol. This new lysosomal membrane carrier system for neutral amino acids resembles the plasma membrane L system in 3T3 Chinese hamster ovary cells and melanoma B-16 cells.
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PMID:Characteristics of a lysosomal membrane transport system for tyrosine and other neutral amino acids in rat thyroid cells. 378 56

DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.
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PMID:Activation of N-ras in a human melanoma cell line. 388 33

Tissue plasminogen activator produced by a human melanoma cell line (Bowes), was purified from large volumes of supernatant fluid using immunosorbent chromatography on monoclonal antibodies, followed by chromatography on lysine-Sepharose 4B and gel filtration on Sephadex G-150. Immunosorbents based on monoclonal antibodies were shown to be preferable to those based on polyclonal antibodies for several reasons: efficient binding and desorption, high yield along with a high degree of purification. The overall recovery was about 50%. A homogeneous sample of single-chain tissue plasminogen activator with a molecular weight of approx. 65 000 was obtained as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Amino acid sequence analysis revealed two N-terminals in equal yields, corresponding to the long and short tissue plasminogen activator structures previously reported by others. The pure preparations of tissue plasminogen activator showed specific activities of about 200 000 IU/mg protein.
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PMID:Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies. 404 Mar 96

Protamine sulfate reversibly inhibits serum-induced mitogenic stimulation of several nontransformed and neoplastic cell types in vitro. Fifty percent inhibition was induced by approximately 120-150 micrograms protamine sulfate/ml. Cells were affected directly, and inhibition depended on the duration of cell exposure. Heparin, chondroitin sulfate, heparan sulfate, and dextran sulfate neutralized protamine sulfate effects during the early stages of treatment. Nontransformed cells [bovine aortic endothelial cells, adult human gingival fibroblasts (strains 423 and 1101), fetal rat skin (strain 921-K) and muscle fibroblasts] required longer exposure to induce inhibition than did neoplastic cells [rat 3-methylcholanthrene-induced fibrosarcoma cell lines (MCA-6 and MCA-9), a macrophage-like cell line (NCTC-3749), Walker 256 rat carcinoma cells (ATCC-CCL-38), rat Morris hepatoma cells (ATCC-CCL-144), murine melanoma cells (B16), and rat bladder squamous cell carcinoma cells (804-G)]. Other polycationic compounds, including histone type VIII-S, poly-L-lysine, poly-L-arginine, and protamine (free base), were also effective inhibitors, whereas the basic proteins cytochrome c and lysozyme had no effect. Poly-L-histidine, poly-L-glutamic acid, poly-L-aspartic acid, and dextran blue also had no inhibitory effect.
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PMID:Protamine sulfate inhibition of serum-induced mitogenic responses: differential effects on normal and neoplastic cells. 621 Mar 90

Previous studies have shown that degradation of the acute phase reactant serum amyloid A (SAA) is mediated by enzymes on the plasma membrane of lymphocytes and monocytes. The responsible enzymes had properties of neutral elastases. The present investigations were conducted to explore whether human NK cells enriched by Percoll gradient centrifugation have similar activity and if so, whether the same or different enzyme classes are responsible for proteolysis as well as for tumor cell lysis. Accordingly, human NK cells were enriched on discontinuous Percoll gradients after which the cells were incubated either with SAA or with [3H] proline-labeled melanoma cells at various effector to target cell ratios. When SAA degradation was followed by SDS-polyacrylamide gel electrophoresis, NK fractions proved to be as effective in digesting the protein as unfractionated mononuclear leukocytes. To characterize the enzymes that may be involved in cytotoxicity on the one hand, and SAA degradation on the other, the NK fractions were treated with the following inhibitors: diisopropylfluorophosphate (DFP), soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethylketone (TLCK), the elastase inhibitors elastatinal, Ac-Ala-Ala-Pro-Val-CH2Cl, Meo-Suc-Ala-Ala-Pro-Val-CH2Cl, and an inhibitor of aryl sulfatase, Na2SO4. Preincubation of the cells with DFP or elastase inhibitors abolished their ability to hydrolyze SAA but did not affect their ability to kill tumor cells. On the other hand TLCK, a potent inhibitor of cytotoxicity, did not bring about any reduction in the proteolysis of SAA. DFP and Na2SO4 diminished cytotoxicity partially. Elimination of NK cells by sorting after incubation of lymphocytes with the monoclonal antisera Leu-7 and Leu-11 abolished cytotoxicity as well as proteolysis. The observations are compatible with the concept that NK cells carry several enzymes with different substrate specificities that may be involved in disparate cellular functions.
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PMID:Different enzyme classes associated with human natural killer cells may mediate disparate functions. 636 42

Different procedures for fixation and processing were evaluated in order to examine the antigenic profile of melanocytes and other epidermal cells for immunoelectron microscopy. For this purpose the monoclonal antibodies anti-HLA-A, B, C, anti-HLA-DR, anti-T6, and the melanoma-associated monoclonal antibody NKI /C-3 were used as markers. Fixation with periodate-lysine-paraformaldehyde yielded better antigenic and ultrastructural preservation than 3% paraformaldehyde or picric acid-paraformaldehyde did. Skin was further processed by five different methods: (a) 15-micron frozen sections; (b) 75-micron agar-embedded, tissue chopper sections; (c) 15-micron polyethylene glycol-embedded sections; (d) epidermal cells in suspension; and (e) epoxy sections (postembedding staining) were prepared for the immunoperoxidase procedure. Antigenicity was best preserved in the cell suspension method and somewhat less, but with a similar staining distribution, with the first three methods. Staining with the polyethylene glycol-embedded sections was only achieved if they were left free-floating in buffer; no staining was observed when the sections were mounted on glass slides and left to dry overnight at 37 degrees C. Epidermal cells remained unreactive in the postembedding method, even after etching. Ultrastructural preservation of the agar-embedded sections and the cells in suspension was superior to the other preembedding methods. Melanocytes mostly showed moderate staining for HLA-A, B, C and slight staining for the antigen that is recognized by NKI /C-3. The latter was further demonstrated on Langerhans cells and indeterminate cells which also expressed HLA-A, B, C, T6, and HLA-DR antigens.
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PMID:Methods in laboratory investigation. Immunoelectron microscopic methods for demonstration of antigens on normal human melanocytes and other epidermal cells. 642 21

The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.
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PMID:Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator. 653 96

Tissue plasminogen activator (t-PA) in plasma was separated from inhibitors by adsorption on lysine-Sepharose. It was then determined indirectly by measuring the plasmin generated from plasminogen with poly-lysine as stimulator, in a chromogenic, parabolic rate assay. The reaction proceeded with tissue plasminogen activator and plasmin(ogen) adsorbed on the gel, and followed the kinetics described for similar parabolic rate assays in soluble systems. The assay was standardized against melanoma plasminogen activator (m-PA) and had the sensitivity range of 0.001-0.020 IU (4-80 pg). Anti-m-PA IgG quenched the activity generated in plasma on venous occlusion and part of the activity in pre-occlusion plasma. The method was sensitive to purified urokinase. The basic plasma values in resting normal individuals were: mean 0.08, range 0.01-0.26 X 10(3) IU/l (n = 19), and after 20 min of venous occlusion: mean 2.48, range 0.24-4.34 X 10(3) IU/l (n = 10). The assay correlates well with a fibrin plate method, r = 0.96.
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PMID:A sensitive assay for tissue plasminogen activator activity in plasma, using adsorption on lysine-sepharose. 654 74

The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine-Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01%) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one-chain form can be proteolytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one-chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N-terminal amino acids. Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, 'A' and 'B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains the active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an epsilon-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form.
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PMID:Purification and characterization of a melanoma cell plasminogen activator. 668 60

Incubation of human Glu-plasminogen with 1,5-difluoro-2,4-dinitrobenzene leads to the specific intra-molecular cross-linking of the kringle 1+2+3 region and the light (B) chain region of plasminogen. This cross-link was found to prevent the conformational change which is induced in Glu-plasminogen by lysine analogues or by proteolytic removal of the NH2-terminal peptide. Our results suggest that the cross-link freezes the closed conformation of Glu-plasminogen, and it seems likely that the transition to the loose conformer requires separation of the kringle 1+2+3 region from the light (B) chain portion. The change in the relative position of these regions during the conformational change in plasminogen is also indicated by our observation that the rate of formation of the intramolecular cross-link is significantly decreased when transition to the loose conformer is induced either by saturation of the lysine-binding sites or by conversion to Lys-plasminogen. Cross-linked Glu-plasminogen is slowly activated by urokinase and melanoma tissue plasminogen activator, but in contrast with uncross-linked Glu-plasminogen conversion to Lys-plasminogen or saturation of lysine-binding sites with ligand does not increase the rate of activation because the cross-link prevents transition to the loose conformer which is susceptible to activation. The fibrin affinity of cross-linked Glu-plasminogen is practically identical with that of Glu-plasminogen. As in the case of uncross-linked Glu-plasminogen, removal of the NH2-terminal peptide causes a marked increase in fibrin affinity although the resulting cross-linked Lys-plasminogen is fixed in the closed conformation. This result suggests that the NH2-terminal peptide inhibits binding of plasminogen to fibrin by direct interaction with the fibrin-binding site, and the conformational change that normally accompanies its removal is not a prerequisite of strong binding.
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PMID:Importance of intramolecular interactions in the control of the fibrin affinity and activation of human plasminogen. 672 59


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