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Results for “label transfer”

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At least 55 records · Page 3Linked to original sources
PubMed1989

Identification of a human protein that interacts with nuclear localization signals.

Through a series of label transfer experiments, we have identified a HeLa cell nuclear protein that interacts with nuclear localization signals (NLSs). The protein has a molecular weight of 66,000 and an isoelectric point of approximately 6. It associates with a synthetic peptide that contains the SV-40 T antigen NLS peptide but not with an analogous peptide in which an asparagine is substituted for an essential lysine (un-NLS peptide). In addition to these peptides, several proteins have been tested as label donors. With the proteins, there is a correlation between nuclear localization (assayed with lysolecithin-permeabilized cells) and label transfer to the 66-kD protein. The NLS peptide (but not the un-NLS peptide) competes with the proteins in label transfer experiments, but neither wheat germ agglutinin nor ATP has an effect. These results suggest that the 66-kD protein functions as an NLS receptor in the first step of nuclear localization. In the course of this work, we have observed that the Staphylococcus aureus protein A is a strongly karyophilic protein. Its dramatic nuclear localization properties suggest that it may have multiple copies of an NLS.

Amino Acid Sequence↗
PubMed1983

[3H]Cholesteryl ester labeling and transfer among human and nonhuman primate plasma lipoproteins.

Aliquots of human and nonhuman primate plasma containing 5,5'-dithiobis (2-nitrobenzoic acid) were incubated at 37 degrees C in tubes previously coated with trace amounts of tritium-labeled cholesteryl oleate ( [3H]CO). Initially, cholesteryl esters were transferred at a rapid rate into plasma after which the rate slowed. During 24 h of incubation, an average of 55% of the [3H]CO transferred from the side of the tube into African green monkey plasma, 44% into human plasma and 21% into rat plasma. Greater than 98% of the radioactive ester transferred into plasma was found to be associated with plasma lipoproteins that were then rapidly separated using vertical rotor density gradient ultracentrifugation. In very low density lipoprotein (VLDL)-poor plasma after 30 min incubations, high density lipoproteins (HDL) contained most of the [3H]CO while 5- to 24-h incubations resulted in increased labeling of low density proteins (LDL). In VLDL-rich plasma, it was found that in addition to the labeling of HDL, VLDL contained about 25% of the labeled cholesteryl esters after 30-min incubations and, as above, the proportion in LDL subsequently increased. Compositional analyses showed that intermediate-sized LDL (ILDL) were accumulating cholesteryl ester mass while transfer occurred. LDL labeled using this method were injected intravenously into monkeys and their removal from plasma was found to be similar to that found for LDL labeled in vivo. It was concluded that this method of plasma lipoprotein cholesteryl ester labeling, presumably a result of cholesteryl ester transfer protein activity, was efficient, resulted in lipoproteins labeled only in the cholesteryl ester moiety, and induced minimal modification of lipoprotein particles that did not alter their biological activity.

Animals↗
PubMed2003

pH- and salt-dependent self-assembly of human Rad51 protein analyzed as fluorescence resonance energy transfer between labeled proteins.

Human HsRad51 protein assembles on a DNA molecule through cooperative binding and forms a long filament for homologous recombination. We have characterized the self-assembly of HsRad51 by measuring the fluorescence resonance energy transfer from the fluorescein-labeled protein to the rhodamine-labeled protein. Self-assembly quickly reached equilibrium and can be described by the head-to-tail polymerization of monomers, like that of its procaryotic homologue, RecA. It depended strongly on pH and was inhibited by high salt concentrations, indicating that ionic interactions between negatively and positively charged aminoacid residues are important. By contrast, neither ATP nor ADP significantly affected the reaction.

DNA-Binding Proteins↗
PubMed1979

Early molecular events in the interaction of enveloped viruses with cells. I. A fluorescence and radioactivity study.

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells. The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell. Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to label transfer from virus to cell membranes or vice versa. While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.

Animals↗
PubMed1995

[Representation and transfer of labeled reference structures in X-ray, CT and MR imaging].

PURPOSE: Image information obtained with different methods should be exactly transferable to profit fully from the single advantage the different imaging method has in localisation and for further treatment planning. Prominent anatomical points serve as reference structures (landmarks) for the transference of imaging information and details of situation. Aim of this study is the improvement of reproductability by comparing topographic relations, measurements and positioning in different imaging-procedures with supporting marking aids/surface markers. MATERIAL AND METHODS: By using pharmacological capsules and plastic tubes filled with oily contrast medium containing iodine (Lipiodol; Byk Gulden), marking aids were developed which can be seen in similar definite limits within the framework of MRI-, CT- and conventional X-ray-Imaging. RESULTS: A method to view these new, artificial markers in combination with individual, anatomical landmarks is introduced. The marking aid/surface marker, fixed on anatomical reference structures on the skin, does not result in an additional burden for the patient. CONCLUSIONS: The new, artificial markers are also useful for making other structures recognizable, such as anatomical relation lines, center of the portal and edges in planning imaging for radiotherapy treatments and are used as leading and reference structures to compare localisation and extent of lesions in X-ray-, CT- and MRI. Marking aids/surface markers do not have to be changed in different imaging methods.

Magnetic Resonance Imaging↗
PubMed2000

Differentiation-dependent cytoplasmic distribution and in vivo RNA association of proteins recognized by the 3'-UTR stability element of alpha-globin mRNA in erythroleukemic cells.

In this study, we analyzed subcytoplasmic distribution and in vivo RNA association of proteins with specific affinity to cytosine-rich stability determinant sequences of alpha-globin mRNA 3'-UTR in a MEL-707 erythroleukemic model. We took advantage of the possibility that these cells can be reversibly differentiated (as a continuous population, but not at the level of individual cells) which, therefore, allows analysis of various stages of erythroid differentiation within the same cell population. Label transfer experiments revealed four major complexes with molecular mass of 110-, 70-, 55- and 50-kDa in various cytoplasmic fractions. Using the combination of in vitro label transfer and in vivo UV-crosslinking techniques, we also demonstrated that subcytoplasmic distribution as well as in vivo RNA association of various complex-forming proteins is differentiation dependent in MEL-707 cells. These results indicate that changes in the cytoplasmic environment imposed by the differentiating stimulus might direct important biochemical signals as to how the stability determinant 3'UTR elements interact with their binding proteins. These data also suggest that stability complexes are dynamic macromolecular structures with high response capacity to various extra- and intracellular regulatory stimuli.

3' Untranslated Regions↗
PubMed2002

Fluorescence labeling methods for microchannel plate capillary electrophoresis DNA sizing.

DNA analysis on microchannel plate (MCP) capillary electrophoresis platforms will be advanced by the development of new methods for the fluorescence labeling of analytes and standard sizing ladders. Here we evaluate end-labeling of commercially available DNA ladders and polymerase chain reaction (PCR) amplicons with terminal deoxynucleotidyl transferase (TdT) as a method for fluorescent double-stranded DNA sizing analysis. A PCR-based procedure for the facile construction of custom energy-transfer-labeled DNA sizing ladders is also presented. High-resolution sizing of single-stranded DNA fragments is demonstrated with this energy-transfer-labeled ladder. These DNA labeling procedures will be useful for double-and single-stranded DNA analyses on microdevices and other electrophoretic platforms.

Base Sequence↗
PubMed1976

[Autographical investigations of the transfer of labeled RNA from macrophages to lymphocytes (author's transl)].

By means of electron microscopic autoradiography we demonstrated a radioactivity in mice spleen lymphocytes after a common cultivation for 1 h with 3H-uridine marked autologian macrophages, washed profoundly. The radioactivity in lymphocytes is small after contact with unstimulated macrophages and is markedly increased after common cultivation with antigen stimulated macrophages. A transfer of RNA molecules of macrophages into lymphocytes is supposed.

Animals↗
PubMed1996

Fluorescently labeled phosphatidylinositol transfer protein isoforms (alpha and beta), microinjected into fetal bovine heart endothelial cells, are targeted to distinct intracellular sites.

Upon permeabilization of Swiss mouse 3T3 fibroblasts, an isoform of phosphatidylinositol transfer protein (PI-TP) was preferentially retained, a major part of which was associated with the perinuclear Golgi system (K. J. de Vries, A. Momchilova-Pankova, G. T. Snoek, and K. W. A. Wirtz, Exp. Cell Res. 215, 109-113, 1994). In the present study, the intracellular localization of this isoform (PI-TP beta) and the regular form (PI-TP alpha) was investigated in fetal bovine heart endothelial cells by microinjection of fluorescently labeled analogs followed by confocal laser scanning microscopy. The PI-TP alpha and PI-TP beta used were purified from bovine brain cytosol and covalently labeled with sulfoindocyanine dyes. By this novel method it was found that PI-TP beta was preferentially associated with perinuclear membrane structures whereas PI-TP alpha was predominantly present in the nucleus and in the cytoplasm. This intracellular localization was confirmed by indirect immunofluorescence indicating that the fluorescently labeled PI-TP alpha and PI-TP beta were targeted to the same sites as their endogeneous counterparts.

Animals↗
PubMed1973

Distribution and blood-to-milk transfer of labeled antibiotics.

RADIOACTIVITY DISTRIBUTION WAS DETERMINED IN SERUM AND MILK OF LACTATING EWES AFTER PARENTERAL ADMINISTRATION OF FIVE LABELED ANTIBIOTICS: (14)C-benzylpenicillin G, (3)H-dihydrostreptomycin, (3)H-tetracycline, (14)C-chloramphenicol, and (14)C-spiramycin. Antibiotic levels were measured simultaneously by microbiological assay. Radiochemical and microbiological assay procedures presented similar kinetic patterns for uptake in serum and penetration into milk, except for tetracycline. Small reductions in milk pH markedly increased the excretion of spiramycin and slightly influenced the milk passage of penicillin, dihydrostreptomycin, and tetracycline but did not alter the transfer of chloramphenicol into milk. Thus, it appears that the five antibiotics penetrate milk in accordance with the nonionic passive diffusion principle, and that good agreement is achieved between the calculated and observed milk/serum ultrafiltrate concentration ratios obtained during equilibrium.

Animals↗
PubMed1999

Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells.

Cytoplasmic proteins with binding capability to AU-rich instability determinant sequences (ARE) of tumour necrosis factor alpha (TNFalpha) mRNA 3' untranslated region (3'UTR) were assessed in human lymphoid cells. In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide-long AUUUA pentamer-containing portion of the 3'UTR had been deleted conferred binding specificity to five major activities of 22/25-, 38/40-, 50-, 60- and 80-kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin-induced disarrangement of the F-actin-based microfilament system led to a Triton X-100-insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV-crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL-2) and TNFalpha mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE-AUBP interactions.

3' Untranslated Regions↗
PubMed1998

Distribution profile and in vivo RNA association of cytoplasmic AU-rich sequence binding proteins in various mammalian cells: effect of the organizational state of cellular architecture.

With the aid of sequential detergent fractionation and label transfer techniques, in this study we monitored the distribution of cytoplasmic AU-rich sequence binding proteins (AUBP) in different cell types. We show here that cells of various origin display diverse AUBP profiles when the presence and abundance of AUBPs were compared in two major cytoplasmic compartments, the non-ionic detergent-soluble and -insoluble fractions. We also demonstrate that different RNA probes derived from AU-rich 3'-UTR elements of various cytokine and proto-oncogene mRNAs detect distinct AUBPs within the same cell. When the in vivo association of these proteins with AU-rich RNA was assessed using the combination of in vivo UV-crosslinking and label transfer methods, we found that detachment of monolayer cells by trypsinization or disruption of the microfilament network by Cytochalasin treatment prior to UV light exposure, led to selective changes in both the cytoplasmic distribution and in vivo RNA association of many AUBPs. These data indicate that the organizational state of cytoarchitecture may influence processes involved in AUBP-mediated mRNA metabolism.

Actin Cytoskeleton↗
PubMed1980

Transfer RNA labeling of Escherichia coli methionyl-tRNA transformylase.

Homogeneous methionyl-tRNA transformylase from Escherichia coli can react with periodate-treated tRNAMetf to form a Schiff's base through a free amino group (probably the epsilon-amino group of a lysine) and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. This reaction is reflected by the loss of activity of the enzyme at saturating tRNA dialdehyde and upon reduction of the Schiff's base by NaBH4. the kinetics of inactivation (37 degrees C, pH 8.5) level off at 50% of the initial enzymic activity. In the presence of 2 mM cyanohydridoborate, a mild reducing agent which leaves intact the reacting aldehyde groups of oxidized tRNA but continuously reduces the Schiff's base in equilibrium, the activity of the enzyme can be destroyed by 100%, at a rate of 0.044 min-1, with the parallel covalent incorporation of close to one tRNA molecule per enzyme molecule. Selectivity of the labeling is also supported by the demonstration that, prior to Schiff's base formation, modified tRNA binds the transformylase with equilibrium constant and stoichiometry in good agreement with those for the active binding of unmodified tRNa. Moreover intact tRNA competes for the inactivation by the dialdehyde derivative with an affinity constant identical to that for its active binding to the enzyme

Acyltransferases↗