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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper describes a method of screening mutagenised populations of an E. coli galA/galB F- prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal+ papillae. With a suitable choice of gal- mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.
Mol Gen Genet 1976 Jan 16
PMID:Isolation of rec- mutants from an F-prime merodiploid strain of Escherichia coli K-12. 76 56

We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.
Mol Pharmacol 1992 Jun
PMID:Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. 135 33

Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vacuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.
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PMID:Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae. 243 74

Fluorescein-isothiocyanate (FITC) labeled lectins were used to study the distribution pattern of specific binding-sites in histological sections of normal and osteoarthrotic articular cartilage from the mouse knee joint. Male inbred mice of the STR/1N-strain develop spontaneous arthrotic articular cartilage lesions on the medial condyle of tibia and femur. The varus-deformity of the knee joint leads to a recurrent medial patellar luxation with osteoarthrotic defects on the medial part of the facies patellaris femoris. It was demonstrated that the lectin staining pattern of osteoarthrotic articular cartilage, especially on the facies patellaris femoris, was different from that of normal articular cartilage. The differences in lectin staining corresponded to those observed between normal and fibrillated articular cartilage from human patellae. The normal articular cartilage of the mouse knee joint possessed lectin binding-sites for Concanavalin A (ConA) and wheat germ agglutinin (WGA), but not for Ulex europaeus agglutinin (UEA), soy bean agglutinin (SBA) and peanut agglutinin (PNA). In addition to the completely changed distribution pattern of ConA and WGA in osteoarthrotic cartilage, SBA, PNA and UEA developed distinct staining patterns particular to the fibrillated areas of arthrotic cartilage. The increased lectin-binding to arthrotic articular cartilage may be due to unmasking of sugars in the course of bondage breakdown in fibrillated cartilage or the production of pathological glycoproteins. It is evident that lectins can demonstrate minute differences between normal and arthrotic cartilage and it is concluded, therefore, that lectins are sensitive and specific tools for the study of degenerative joint diseases.
Virchows Arch B Cell Pathol Incl Mol Pathol 1988
PMID:Lectin-binding in normal and osteoarthrotic articular cartilage from STR/1N-mouse knee joints. 289 40

The mechanisms of allosteric regulation of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum by ATP have been compared. Although both enzymes showed stimulation of ATPase activity by ATP, the cardiac enzyme did not show the plateau in ATPase activity at 10-100 microM ATP seen with the skeletal enzyme. Likewise the phosphoenzyme (EP) levels did not plateau with the cardiac enzyme as they did with the skeletal enzyme. The apparent negative cooperatively which was seen in the kinetics of ATP hydrolysis at low ATP concentrations was not due to negative cooperatively in substrate binding to either enzyme. The cardiac enzyme did show, however, much higher affinity for the ATP analog, AMPPCP, which helps explain how AMPPCP blocks ATPase activity in the cardiac enzyme and stimulates ATPase activity in the skeletal enzyme. Fluorescein isothiocyanate was used to determine if allosteric regulation takes place through site-site interactions in oligomers. The 1 to 1 ratio between AMPPCP binding sites and FITC binding sites eliminated allosteric regulation by effector sites in both enzymes. The allosteric mechanism which remained was one in which the active-site becomes an effector-site by the early departure of ADP in the reaction mechanism. The step stimulated by the binding of ATP at the active-site turned effector-site was a nonphosphorylated form of the enzyme in cardiac sarcoplasmic reticulum and a phosphorylated form in skeletal sarcoplasmic reticulum.
Mol Cell Biochem
PMID:Allosteric regulation of cardiac sarcoplasmic reticulum Ca-ATPase: a comparative study. 297 11

Fluorescein esters are employed in assays of cell viability, membrane permeability and esterase activity. The ester most widely used, fluorescein diacetate (FDA), has the disadvantage of rapid cellular efflux of its hydrolysis product fluorescein. This is particularly problematic for flow cytoenzymology (FCE), where fluorescence is measured in individual cells allowing identification of subpopulations differing in esterase activity and/or membrane characteristics. We present a comparison of FDA with two potentially improved substrate probes for FCE, carboxyfluorescein diacetate (CFDA) and bis(carboxyethyl)-carboxyfluorescein-tetra acetoxy methyl ester (BCECF-AM). Substrates were characterized in terms of reaction and product efflux kinetics in EMT6 mouse mammary tumour cells, together with inhibition kinetics for the carbamoylating agent BCNU. Intact viable cells were analysed by FCE and spectrofluorimetry, and the latter was also used for cell sonicates and purified esterase. CFDA and BCECF-AM enter cells and are hydrolysed more slowly than FDA. CFDA and FDA hydrolyses obey Michaelis-Menten kinetics with Km values of around 19 and 2 microM, respectively, whereas BCECF-AM hydrolysis deviates from this classical behaviour. BCNU (5 X 10(-4) M) inhibits FDA and BCECF-AM hydrolyses by approximately 50%, compared to 30% for CFDA. CFDA may be partly hydrolysed by membrane-bound esterases. Efflux half-lives were 16 min, 94 min and greater than 2 h for products of FDA, CFDA and BCECF-AM, respectively. We conclude that BCECF-AM is the optimal substrate probe for FCE. This study emphasizes the need to optimize various parameters when selecting a substrate for flow cytoenzymological assay or when loading other reporter fluorochromes into cells via lipophilic esters.
Mol Cell Probes 1988 Jun
PMID:Polar fluorescein derivatives as improved substrate probes for flow cytoenzymological assay of cellular esterases. 317 58

The binding of the plant toxin ricin to various life cycle stages of Schistosoma mansoni has been studied. Fluorescein isothiocyanate (FITC)-ricin exhibited binding to schistosomula and adult worms, but not to cercariae or to freshly transformed schistosomula. Binding was specifically inhibited by the presence of galactose in the medium. Analysis of protein synthesis in treated parasites illustrated that ricin failed to intoxicate schistosomula or adult worms. Indeed, fluorescence quenching studies suggested that the fluorescent toxin was confined to the outer bilayer and was not internalised.
Mol Biochem Parasitol 1987 May
PMID:Interaction of the plant toxin ricin with different life cycle stages of Schistosoma mansoni. 361 73

Fluorescein-conjugated monoclonal antibodies were used to investigate the properties of the Lyt-1, Lyt-2 and Lyt-3 antigens on the murine lymphocyte cell surface. Monoclonal antibody to Lyt-1 patched and capped on the thymocyte cell surface, whereas an Fab fragment of the antibody gave a uniform distribution of fluorescence on most cells and diffused freely in the plane of the membrane. Fluorescence photobleaching measurements showed that the intact antibody was relatively immobile on the cell surface, whereas the Fab fragment was freely mobile. Monoclonal antibody to Lyt-2 or Lyt-3 (which are linked to one another by disulfide bonds) gave a uniform distribution of fluorescence and was mobile on the cell surface. When cells were incubated with antibodies to Lyt-2 and Lyt-3 simultaneously, cells appeared patched and the lateral mobility of the antibodies was greatly reduced. Treatment of cells with either sodium azide or 2-mercaptoethanol followed by N-ethyl maleimide to reduce and block sulfhydryl groups inhibited the patch formation caused by simultaneous incubation with both Lyt-2 and Lyt-3 antibodies.
Mol Immunol 1982 Nov
PMID:The lateral mobility and surface distribution of Lyt-1, Lyt-2 and Lyt-3 on mouse thymocytes. 619 Dec 1

Fluorescein isothiocyanate-conjugated lectins (FTC-lectins) have been used to study the occurrence and the distribution of their receptors in the coating of mouse myocardial cells throughout their differentiation (from 10 days post-coitum to the adult stage). FTC-Con A, -WGA and -PNA specifically inhibited by alpha-mannosyl, beta-N-acetylglucosaminyl and beta-galactosyl residues, respectively, bind to the cell surface whatever the stage of differentiation. FTC-SBA and -HPA, specifically inhibited by alpha-N-acetylgalactosaminyl residues, bind to the coating of adult cells; their receptor sites appear only at day 18 of development. FTC-UeA, specifically inhibited by fucosyl residues also binds to adult cells. In sections of embryonic hearts, FTC-UeA is trapped by a lectin-like component; this UeA binding can be prevented with mannosyl-substituted serum albumin. Isolated embryonic myocardial cells do not bind FTC-UeA. FTC-BS I-A4 and -BS I-B4, specifically inhibited by alpha-N-acetylgalactosaminyl and alpha-galactosyl residues, respectively, bind only to the capillaries of adult heart. These results show that the differentiation of myocardial cells is accompanied by the sequential acquisition of lectin-binding sites. These changes express the maturation of cell coating glycoconjugates.
J Mol Cell Cardiol 1983 Feb
PMID:Evolution of the surface of mouse myocardial cells during ontogenesis. II. Changes of fluorescent lectin-binding sites. 634 20

Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.
Mol Immunol 1982 Jul
PMID:Biotinylation of human C3. 712 68


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