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Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes (1982)

Painter, Richard G. ; Schmitt, Manfred ; Jesaitis, Algirdas J. ; [et al.]

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 20 (1982), S. 203-214

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ISSN: 0730-2312

Keywords: N-formyl peptide receptor ; photoaffinity labeling ; polymorphonuclear leukocytes ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 ∼ 0.7 nM). When incubated at 0°C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 Å on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcb.240200211

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The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies (1982)

Jesaitis, Algirdas J. ; Naemura, Joseph R. ; Painter, Richard G. ; [et al.]

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 20 (1982), S. 177-191

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ISSN: 0730-2312

Keywords: N-formyl-chemotactic peptide ; granulocytes ; subcellular fractionation ; peptide receptors ; endocytosis ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4°C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[l25I] Tyr-N°(6-(4′-azido-2′-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37°C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4°C or incubated at 37°C for 2 min or less. Fractionation of cells incubated at 37°C for longer times revealed that the radioactivity sedi-mented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37°C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% ± 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction consedimenting with dense granules.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcb.240200209

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Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast killer virus K28 (1992)

Schmitt, Manfred J. ; Tipper, Donald J.

New York, NY [u.a.] : Wiley-Blackwell

Yeast 8 (1992), S. 373-384

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ISSN: 0749-503X

Keywords: Yeast ; double-stranded RNA virus ; K28 killer virus ; Life and Medical Sciences ; Genetics

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology

Notes: The killer phenotype expressed by Saccharomyces cerevisiae strain 28 differs fron that of the more extensively studied K1 and K2 killers with respect to immunity, mode of toxin action and cell wall primary toxin receptor. We previosly demonstrated that the M28 and L28 dsRNAs found in strain 28 are present in virus-like particles (VLPs) and that transfection with these VLPs is sufficient to confer the complete K28 phenotype on a dsRNA-free recipient cell. We also demonstrated that L28, like the L-A-H species in K1 killers, has [HOK] activity required for maintenance of M1-dsRNA, and predicted that M28 would share with M1 dependence on L-A for replication. We now confirm this prediction by genetic and biochemical analysis of the effects of representative mak, ski and mkt mutations on M28 maintenance, demonstrating that M28 replication resebles M1 in all respects. We also show that L28 is an L-A-H species lacking [B] activity, and that M28 excludes both M1 and M2 from the same cytoplasm. Stable coexpression of K28 phenotype from M28 and of K1 phenotype from an M1-cDNA clone was demonstrated. Exclusion, therefore, acts at the level of dsRNA replication, presumably reflecting competition for the L-A-H encoded capsid and cap-pol fusion protein, rather than reflecting incompatibility of toxin or immunity expression. Finally, we show that expression of active K28 toxin, bu t not of K28 immunity, requires the Kex2 endoprotease.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/yea.320080505

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Expression of the human urokinase-type plasminogen activator receptor in E. coli and Chinese hamster ovary cells: Purification of the recombinant proteins and generation of polyclonal antibodies in chicken (1995)

Magdolen, Viktor ; Rettenberger, Peter ; Lopens, Antje ; [et al.]

Weinheim : Wiley-Blackwell

Electrophoresis 16 (1995), S. 813-816

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ISSN: 0173-0835

Keywords: Affinity chromatography ; Microsequencing ; Chicken antibodies ; Urokinase receptor ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology

Notes: The receptor for urokinase-type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase-type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1-284 of human uPAR) was expressed with an N-terminal histidine-tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1-277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Prior to immunization the N-termini of the recombinant uPAR variants were determined by amino acid sequence analysis. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.

Additional Material: 3 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/elps.11501601134

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Mutational analysis of the genes encoding urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in advanced ovarian cancer (1997)

Türkmen, Beyhan ; Schmitt, Manfred ; Schmalfeldt, Barbara ; [et al.]

Weinheim : Wiley-Blackwell

Electrophoresis 18 (1997), S. 686-689

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ISSN: 0173-0835

Keywords: Sequence variations ; Urokinase ; Urokinase-type plasminogen activator receptor ; Polymorphism ; Restriction fragment length polymorphism ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Evidence has accumulated that urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1) and receptor (uPAR) are involved in tumor invasion and metastasis. We analyzed the DNA sequences encoding these factors to see if they are altered in the ovarian cancer cell lines OV-MZ-6, OV-MZ-19, and OVCAR-3. In the uPA-encoding cDNA derived from OV-MZ-6 cells (but not in the uPA-cDNA from OVCAR-3 and OV-MZ-19), a so-far unknown mutation was identified in codon 121, resulting in a proline to leucine exchange. This exchange creates an AluI restriction site making restriction fragment length polymorphism (RFLP) analyses possible. Previously published PAI-1 sequences pointed to a variation of amino acid 15 of the PAI-1 signal sequence representing either threonine or alanine, which was confirmed in the present study. The uPAR cDNAs of all three cell lines encoded the published wild-type sequence. In order to elucidate the possible role of the Pro121Leu exchange in uPA and the Ala/Thr variants in the signal sequence of PAI-1 in the development and/or progression of human ovarian cancer, we studied the presence of these mutants or variants in a series of 22 ovarian cancer tissues. In addition to the wild-type sequence, the Pro121Leu exchange in the uPA sequence was detected in 10 out of 22 tumor tissues; 11 tumors carried exclusively the Pro121 allele; in one case exclusively the Leu121 allele was detected. In 18/22 tumors, triplet 15 in the signal sequence of PAI-1 encoded alanine, four DNAs contained both the Ala and the Thr allele. Furthermore, we analyzed another known common single-base-pair insertion/deletion polymorphism (ins/del allele) found in the promoter region of the PAI-1 gene and thought to be of functional importance in regulating PAI-1 gene expression. The PAI-1 ins-allele was found in 3/22, the del-allele in 6/22 and both alleles in 13/22 ovarian cancer tissues. In genomic DNA isolated from peripheral blood of 23 healthy donors, we observed similar allele frequencies of the three polymorphisms as found in the 22 ovarian carcinomas. Taken together, these results suggest that the polymorphisms observed in the uPA and PAI-1 genes may not be linked to ovarian cancerPresented at the “Elektrophorese Forum “96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23-25, 1996..

Additional Material: 2 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/elps.1150180505

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