Biomedical subjects

C W Turck

Publications and source records attributed to C W Turck.

107 records · Page 6Linked to original sources

PubMed1989

Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase.

A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase. The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids. A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase. The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies. Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase. Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase. The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence. However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases. These data suggest that cholesterol esterase may be a member of the serine esterase supergene family. Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein. This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo. Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells.

Amino Acid Sequence↗

PubMed1989

Preferential cleavage of amino- and carboxyl-terminal oligopeptides from vasoactive intestinal polypeptide by human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11).

Human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) cleaved synthetic vasoactive intestinal peptide (VIP1-28) with time-and peptidase concentration-dependence, which left less than 30% intact after 30 micrograms was incubated at 37 degrees C with 0.1 micrograms and 10 micrograms of peptidase for 120 min and 15 min, respectively. The rank order of relative rates of peptidolysis amino-terminal to hydrophobic amino acids was Ala4 and Val5 greater than Tyr22 and Ile26 much greater than Leu13 and Met17. The many effects of VIP1-28 on epithelial cell and leukocyte functions thus may be influenced by degradation of the mediator by enkephalinase at the surface of target cells.

Glycopeptides↗

PubMed1989

Diverse responses of human fibroblasts to a highly purified fibroblast-activating factor from the U937 line of human monocytes.

The responses of human dermal fibroblasts to highly purified fibroblast-activating factor (FAF), derived from supernatants of U937 human monocytes stimulated with phorbol myristate acetate (PMA), were investigated with several in vitro assays of specific synthetic functions. The highly purified peptide was detected as a single 16,000-18,000 MW protein, by both silver staining and Western blot analysis, with an antiserum generated against a synthetic peptide representing the amino-terminal sequence of 17 amino acids. At concentrations that induced similar levels of fibroblast proliferation, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1), acidic fibroblast growth factor (aFGF) and FAF also stimulated fibroblasts to generate and release prostaglandin E2 (PGE2) and proteoglycans. TGF-beta had the least effect on proteoglycan production. In contrast, the production and secretion of collagen evoked by FAF was only minimal when compared to that observed with IL-1 and aFGF. FAF and aFGF promoted fibroblast-induced collagen gel contraction with similar potency. Thus, the profile of fibroblast effects is a specific property of each cytokine.

Antigens, Neoplasm↗

PubMed1989

Unique pattern of cleavage of vasoactive intestinal peptide by human lymphocytes.

Human cultured T lymphocytes of the Jurkat line and myeloma cells of the U266 line cleaved the 28 amino acid vasoactive intestinal peptide (VIP1-28) preferentially at three sites with time- and temperature-dependence. The fragments VIP4-28 and VIP23-28) from an endopeptidase activity, and VIP15-28 from a trypsin-like peptidase, together represented a range of 26-65% of the VIP1-28 recovered after 2 hr at 37 degrees C or 4 hr at 22 degrees C, based on the absorbance of purified peptides and the radioactivity of [125I]Tyr10 VIP1-28. The endopeptidase activity was associated with membranes recovered after disruption of U266 cells by nitrogen cavitation. Pretreatment of intact U266 and Jurkat cells with diisopropylfluorophosphate (DFP) and the subsequently isolated subcellular particles with phenylmethylsulphonylfluoride (PMSF) and leupeptin inhibited the trypsin-like enzyme by a mean of 80%, without suppressing endopeptidase activity. In contrast, 0.1 mM DL-thiorphan and phosphoramidon blocked selectively a range of 35-70% of the endopeptidase activity in membrane preparations and intact cells. The capacity of lymphocytes to degrade VIP1-28 may substantially alter the effects of this neuromediator on functions of some subsets of T and B cells.

B-Lymphocytes↗

PubMed1988

Isolation and partial characterization of a fibroblast-activating factor generated by U-937 human monocytic leukocytes.

Human cultured monocyte-like tumor cells of the U-937 line, that are incubated for 24 h at 37 degrees C with 100 ng/ml of PMA, generate and release a fibroblast-activating factor (FAF) capable of enhancing the uptake of [3H]TdR by human dermal fibroblasts in vitro. The predominant FAF activity in unpurified supernatants from U-937 cells was associated with one protein of 16 to 18 kDa, as assessed by SDS-PAGE and pI 4 to 5, as determined by IEF in gel. Isolation of the FAF in 15-liter batches of supernatant from cultures of 1.5 x 10(10) PMA-stimulated U-937 cells by ammonium sulfate precipitation and sequential filtration on Sephadex G-50, anion exchange chromatography, and reversed phase HPLC yielded microgram quantities of a homogeneous protein of sufficient purity for structural studies. Purified FAF was not absorbed by affinity columns bearing antisera to IL-1 beta, TNF or basic fibroblast growth factor die not share any tryptic peptides with IL-1 alpha or acidic fibroblast growth factor when analyzed by two-dimensional electrophoresis/chromatography on cellulose thin-layer plates, and has an amino-terminal sequence of amino acids that is different from any known fibroblast growth factor. FAF thus represents a unique human monocyte-derived protein that selectively stimulates human fibroblast proliferation and other functions.

Cell Line↗

PubMed1988

Structurally distinctive vasoactive intestinal peptides from rat basophilic leukemia cells.

Peptides recognized by rabbit antibodies to vasoactive intestinal peptide (VIP) were extracted from diisopropyl fluorophosphate-treated rat basophilic leukemia (RBL) cells and resolved by filtration on Sephadex G-25 in 50 mM acetic acid. The immunoreactive VIPs of RBL cells eluted from Sephadex G-25 at 35-41%, 53-60%, and 69-73% bed volume, but not at 63-68% as for the neuropeptide VIP1-28. The two forms of immunoreactive VIP larger than VIP1-28 reacted with antibodies to both VIP1-9 and VIP10-28, but the smallest was bound only by antibodies to VIP10-28. The smallest immunoreactive VIP was purified by ion-exchange and reverse-phase high-performance liquid chromatography, and the amino acid sequence was determined to be that of VIP10-28 with asparagine-free acid at the carboxyl terminus rather than the amide of VIP neuropeptide. Challenge of RBL cells with 1 microM ionophore A23187 at 37 degrees C released VIP10-28 rapidly to a mean of 75% at 5 min and 77% at 30 min. The VIP generated and released by mast cells thus consists of a mixture of peptides that all differ structurally from the neuropeptide VIP.

Amino Acid Sequence↗

PubMed1987

Selective localization of vasoactive intestinal peptide and substance P in human eosinophils.

Extracts of purified human eosinophils had a mean concentration of 72 fmol of immunoreactive vasoactive intestinal peptide and 21 fmol of substance P per 10(7) eosinophils, that were significantly higher than the content of immunoreactivity of the same neuropeptides in neutrophils, mononuclear leukocytes, and platelets. In contrast, the lower concentrations of calcitonin gene-related peptide and somatostatin were similar in extracts of all leukocytes. Chromatography of the peptides from eosinophils confirmed their identity with vasoactive intestinal peptide and substance P from neuroendocrine sources. Stores of some neuropeptides may endow eosinophils with unique roles in host defense and hypersensitivity reactions.

Chromatography, High Pressure Liquid↗

PubMed1987

Immunological mediators of wound healing and fibrosis.

T-lymphocytes, monocytes, and macrophages, which are the central constituents of immunological and chronic inflammatory reactions, generate numerous polypeptides and other factors capable of stimulating and modulating the proliferation and functions of fibroblasts. These principles differ widely in structure, target cell preference, and functional specificity. The involvement of immunological mediators of fibroblast activities in normal wound healing has not been defined, but a role in some chronic fibrosing disorders, including rheumatoid arthritis, has been suggested by the findings of functionally relevant concentrations in affected tissues. The elucidation of both the pathways of production of fibroblast-activating factors (FAFs) and the determinants of fibroblast responses will permit new approaches to the diagnosis and treatment of deficiencies in wound healing and of abnormal fibrosis.

Antigens, Neoplasm↗

PubMed1986

Structural analyses of a monoclonal heterodimeric suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10.

A GAT-specific, MHC-restricted "second-order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 was biosynthetically radiolabeled with 35S-methionine and was isolated from cell extracts. The isolation procedure involved two-dimensional nonreducing/reducing SDS-PAGE and electroelution of the reduced off-diagonal polypeptide chains from the gel. Biochemical characterization studies revealed that TsF2 is a disulfide-linked heterodimer composed of a basic and an acidic polypeptide chain, both having m.w. of 30,000. Both chains are glycosylated and contain sialic acid residues. The basic polypeptide reacts with anti-I-J antisera, whereas the acidic chain contains the antigen-binding capacity. Monoclonal antibodies induced by immunizing rats with TsF2 purified from hybridoma supernatants were selected for the ability to block immunosuppression mediated by TsF2 in vitro. These antibodies, but not irrelevant antibodies, immunoprecipitated the 35S-methionine-labeled protein that migrates off the diagonal in two-dimensional gels. Thus, we have verified that the immunosuppressive protein that migrates off the diagonal in two-dimensional gels binds to antibodies that are known to inhibit the biologic activity of unpurified TsF2.

Animals↗

PubMed1985

Purification and partial characterization of a monoclonal "second order" suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10.

A GAT-specific "second order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 has been purified and biochemically characterized. The protein has a m.w. of approximately 66,000, an isoelectric point of 6.8 to 6.9, and elutes from a reversed phase HPLC column in two peaks, one in 55% acetonitrile, the other in 70% propanol. Amino acid analysis of both forms gave similar molar ratios, suggesting that the two forms are closely related and may differ mainly in the degree of posttranslational modification. SDS-PAGE electrophoresis under reducing conditions gave two chains of the apparent m.w. of 42,000 and 35,000.

Animals↗

PubMed1981

[Synthesis of defined peptide derivatives via aminolysis of 3-[N alpha-acylpepidyloxy)-2-hydroxy-N-alkylbenzamides at elevated temperatures, I. Synthesis of the model peptide derivative Z-Ala-Phe-Gly-N(Et)2 (author's transl)].

Reacting 3-(N alpha-Benzyloxycarbonylalanyl-phenylalanyloxy)-2-hydroxy-N-methylbenzamide with glycinediethylamide at temperatures within 60-100 degrees C, utilizing either dimethylsulfoxide, dimethylformamide or N-methylpyrrolidone as solvents, yielded the corresponding tripeptide derivative Z-Ala-D/L-Phe-Gly-N(Et)2. Depending on the reaction temperature and the nature of the solvent, the isolated tripeptide derivatives contained 0.55% (60 degrees C, dimethylformamide) to 2.5% (100 degrees C, dimethylformamide) of the D-epimer, which could be separated by high-performance liquid chromatography. 3-(N alpha-Benzyloxycarbonylpeptidyloxy)-2-hydroxy-N-methylbenzamide derivatives allow peptide couplings at elevated temperatures even in those cases where the poor solubility of the amino components prevents the condensation step with those methods operating at low temperatures.

Benzamides↗

PubMedDate not supplied

Identification and sequence of a fourth human T cell antigen receptor chain.

Thymus-derived lymphocytes (T cells) use clonally distributed antigen receptors to recognize peptide fragments associated with products of the major histocompatibility complex (MHC) (refs 1-4). On most murine and human T cells the T cell receptor (TCR) is composed of disulphide-linked alpha and beta chains (TCR alpha/beta), each of which contains constant and variable domains, and which are associated with the invariant chains of the CD3 complex. It has been demonstrated, however, that a distinct CD3-associated TCR is expressed on a small subset of T cells or immature thymocytes which fail to express either CD4 or CD8 (refs 7-14), the molecules associated with class II or class I MHC antigen recognition. Instead of TCR alpha/beta, these cells express heterodimers of gamma and delta chains (TRC gamma/delta). The genes encoding alpha, beta, and gamma have been isolated and characterized. A new murine T cell receptor (Cx) gene which undergoes rearrangement and expression early during T cell ontogeny has recently been identified 5' of the murine J alpha C alpha gene locus. Here we isolate and sequence the homologous transcript from PEER, a human cell line that expresses a TCR gamma/delta, and show that it encodes a protein with characteristic V, D, J, and C segments. Using probes derived from this transcript, we have shown that both PEER and MOLT-13, another TCR gamma/delta-expressing cell line, rearrange this locus and express two sizes of transcripts differing in the 3' untranslated region. Using a synthetic peptide derived from the deduced C region sequence, we have prepared antisera that precipitates the delta chain of the TCR from both PEER and MOLT-13, thus demonstrating that Cx and its human homologue code for the delta chain of the TCR.

Amino Acid Sequence↗

PubMedDate not supplied

Apolipoprotein E-binding proteins isolated from dog and human liver.

Chylomicron remnant catabolism appears to be mediated by apolipoprotein (apo) E binding to hepatic lipoprotein receptors. Previously, the apo B,E(LDL) receptor and a unique apo E-binding protein (referred to as the apo E receptor) were isolated from solubilized canine and human livers. In the present study, the apo E-binding fraction was further characterized and found to contain at least three proteins, all of which bind apo E-containing lipoproteins with high affinity. The 56-kDa band was found to contain the alpha- and beta-subunits of F1-ATPase, presumably derived from mitochondrial membranes. In addition, an apo E-binding protein with an apparent Mr approximately equal to 59,000 was identified. The 59-kDa protein displays calcium-independent binding on ligand blots, but displays both calcium-dependent and -independent binding in assays performed with detergent-solubilized protein. The 59-kDa protein recognized lipid-free as well as lipid-bound apo E in ligand blots, and also bound apo E-2, apo E-3, and apo E-4 in a comparable way. Monoclonal antibodies produced against the 59-kDa protein did not react with the 56-kDa proteins. Normal human liver, as well as the liver of a patient lacking the apo B,E(LDL) receptor, possessed the 56-kDa and 59-kDa proteins. These data indicate that liver cells possess at least three proteins, in addition to the apo B,E(LDL) receptor, that bind apo E-containing lipoproteins with high affinity. The physiological role of these proteins in apo E metabolism remains to be determined.

Animals↗

PubMedDate not supplied

Identification of phosphotyrosine residues in peptides by high performance liquid chromatography on-line derivative spectroscopy.

A method has been developed for the detection of phosphotyrosine residues in peptides based on reversed phase-high performance liquid chromatography (RP-HPLC) on-line spectral analysis. It has been found that tyrosine-containing peptides show a hypsochromic shift of the aromatic absorbance maximum when the tyrosine is phosphorylated. Subsequent second-order derivative spectra likewise reveal a hypsochromic shift of the corresponding minima of the phosphotyrosine residues compared to the unmodified tyrosine. This method allows mapping of tyrosine phosphorylation sites in proteins after cleavage into smaller peptides and separation and on-line spectral analysis of the latter by RP-HPLC. It furthermore provides a useful way for the characterization of synthetic phosphotyrosine-containing peptides.

Amino Acid Sequence↗

PubMedDate not supplied

Identification of phosphotyrosine residues during protein sequence analysis.

Synthetic tyrosine-phosphorylated peptides were subjected to protein sequence analysis using a gas-phase sequencer and on-line phenylthiohydantoin (PTH) amino acid analysis. Our data show that phosphotyrosine is stable to the gas-phase sequencing chemistry and can be detected as its PTH-derivative during routine sequence analysis without the need of prior tyrosine radiolabeling.

Amino Acid Sequence↗

PubMedDate not supplied

Mapping of tyrosine kinase autophosphorylation sites with synthetic peptide substrates.

Tyrosine phosphorylation is a key step in the regulation of many cellular events including signal transduction of stimulated growth factor receptor tyrosine kinases. Upon ligand activation, these proteins undergo dimerization and subsequent auto- and transphosphorylation events on specific tyrosine residues, which enables them to interact with several cellular signaling proteins. We have used synthetic peptides encompassing all the tyrosine residues of a tyrosine kinase and employed them as substrates in in vitro kinase reactions. Using this assay we have shown that short tyrosine-containing peptides derived from the cytoplasmic domain of the mouse platelet-derived growth factor beta receptor (PDGF-R) can serve as specific phosphorylation targets for the kinase. These peptides include 7 out of 8 tyrosines that are known auto- or transphosphorylation sites in vivo, as previously determined by peptide mapping and mutational analyses. We have also identified 10 additional tyrosine-containing peptides that are phosphorylated and represent possible novel auto- or transphosphorylation sites of PDGF-R. The presented method greatly simplifies the mapping of auto- or transphosphorylation sites in tyrosine kinases and provides a valuable tool in the analysis of signaling mechanisms involving these proteins.

Amino Acid Sequence↗

PubMedDate not supplied

Screening of synthetic peptide libraries with radiolabeled acceptor molecules.

A method has been developed for the identification of specific acceptor molecule-binding sequences from a chemically synthesized peptide library. The peptide resin beads, each bead carrying one peptide sequence, are incubated with radiolabeled acceptor molecule and subsequently immobilized in a thin layer of agarose. Resin beads that carry acceptor molecule-specific sequences are identified by autoradiography and subjected to automated gas-phase sequencing. The method was tested by screening a synthetic pentapeptide library with an anti-beta-endorphin monoclonal antibody.

Amino Acid Sequence↗