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At least 631 records · Page 35Linked to original sources
PubMed1981

Amino acid sequence adjacent to a sulfhydryl group exposed on illumination of bovine rhodopsin.

Two sulfhydryl groups of bovine rhodopsin, available for chemical modification only after bleaching, were specifically labeled with radioactive iodoacetamide. The labeled protein was extensively reduced and alkylated and digested with pronase, and peptides were purified by gel filtration chromatography, anion and cation exchange chromatography, and high pressure liquid chromatography. Purified peptides were detected by their radioactivity, UV spectral properties, and by their fluorescence after reaction with fluorescamine. Sequence analysis of a highly purified peptide established the sequence S-carboxamido[14C]methyl cysteinyl-prolyl-glycine as the site of one of the light-exposed sulfhydryl groups of bovine rhodopsin.

Amino Acid Sequence↗
PubMed1980

Change of polyamine content in mouse skin by leupeptin, a protease inhibitor, during early stage of tumorigenesis.

The effect of a microbial protease inhibitor, leupeptin, on the content of polyamine in the mouse skin was examined during the early stage of tumorigenesis induced by a single application of 7,12-dimethylbenz[a]anthracene (DMBA) and repeated application of croton oil thereafter. Polyamine content in the skin was measured at 3, 5, 7, and 9 weeks during tumorigenesis. The mice with no visible tumor were selected for measurement of polyamine content at 9 weeks. Mice were left untreated for at least 1 week before measurement of polyamine. Polyamine in the skin was extracted with ice-cold 0.4N HClO4 and separated into putrescine, spermidine, and spermine fractions through CM-cellulose column. Polyamine concentration was determined by fluorometry with fluorescamine. Group A mice painted with croton oil 3 times a week did not develop tumors. Group B mice painted with a single DMBA developed skin tumors, and group C mice painted with a single DMBA and croton oil 3 times a week showed higher development of skin tumors than group B. Group D mice treated as group C and then painted with leupeptin about 2 hr after croton oil treatment. Animals in groups B, C, and D had higher spermidine content as group A at 3 and 5 weeks. Content of spermidine in group B decreased at 7 and 9 weeks compared with group C which had a high content throughout the time tested. Leupeptin treatment in group D inhibited spermidine content in the skin after 7 weeks without affecting until 5 weeks.

Animals↗
PubMedDate not supplied

Spectrofluorimetric techniques in the study of the interaction between fluorescent dyes and proteins or nuclear acids.

After a review of the applications of spectrofluorimetric techniques to the study of ligand--biological substrate interactions, mention is made of the main fluorescent dyes used for the demonstration of structural characteristics and conformational alterations in proteins, nucleic acids, etc. The possibilities of using new compounds recommended in the literature (fluorescamine, fluoropa, a.o.) for the identification and study of proteins are analysed and original investigations with newly synthesized fluorescent compounds (anthraquinone dyes, anhydrides of peridicarboxylic acids, Tb3+ and Sm3+ complexes of the pyridine -2,6-dicarboxylic acid) are presented.

Chemical Phenomena↗
PubMedDate not supplied

[Modification of the platelet cell membrane and suppression of its function with sodium hypochlorite, detected by optical methods].

The platelet release reaction induced by thrombin was studied by means of the kinetic fluorometric method with usage of the fluorescent probe, acridine orange, and the platelet aggregation was measured with Born's turbidometric method. The release reaction and the arachidonic acid peroxidation are suppressed by sodium hypochlorite at the low concentrations (10-20 mcM) to a greater extent than the platelet aggregation. It is possible that such the inhibitory action of sodium hypochlorite is due to a membrane modification which leads to an alteration of intracellular signalling. Amino groups of platelet plasma membrane estimated by means of the fluorescamine fluorescent label are significantly destroyed at the high concentrations of sodium hypochlorite when the strong inactivation of platelets are observed.

Animals↗
PubMed1995

Activation of intracellular proteases is an early event in TNF-induced apoptosis.

The serine protease inhibitor tosyl-argenine methyl ester inhibits TNF-induced apoptosis, suggesting that proteolysis is necessary for this response. To test this hypothesis, we asked whether protein fragmentation occurs during the death of C3HA fibroblasts, a 3T3-like cell that was rendered sensitive to TNF by cycloheximide. Our results show that the binding of fluorescamine, which binds primary amines, was increased in apoptotic cells by approximately 50%. We also found that 10-15% of the protein in apoptotic cells was no longer precipitable by TCA. Evidence for proteolysis was also revealed by SDS-PAGE analysis and from Western blots. We observed fragmentation and/or degradation of lamin B, topoisomerase I, histone H1, protein kinase C beta 1, and cPLA2, indicating that proteolysis during apoptosis is non-specific. We also found evidence of proteolysis in C3HA cells sensitized to TNF by the adenovirus dl758 (which lacks the E3 14.7-kDa resistance gene) suggesting that protease activation is common in TNF-induced apoptosis. In contrast, the adenovirus E3 14.7-kDa resistance gene prevented proteolysis suggesting that this protein acts at, or upstream of the proteases activated in this response. Finally, because tosyl-argenine methyl ester inhibits the release of [3H]arachidonic acid from apoptotic cells, we tested whether proteolysis of cPLA2 is necessary for enzyme activation. Our results failed, however, to reveal a common proteolytic fragment in different cell types, and when tested in vitro the cytosol from apoptotic cells had less cPLA2 activity. It is unlikely, therefore, that proteolysis is necessary for the activation of this enzyme during TNF-induced apoptosis.

Adenovirus E1A Proteins↗
PubMedDate not supplied

Screening of chloramphenicol residues in pork muscle by planar chromatography.

A rapid planar chromatographic method is described for the qualitative determination of chloramphenicol residues in muscle. The drug is extracted with ethyl acetate and purified by solid-phase extraction. After elution, the collected phase is evaporated to dryness. The residue is dissolved in methanol, spotted on a silica gel plate, and chromatographed. After elution, the chloramphenicol is visualized after reduction with fluorescamine. This method detects chloramphenicol in meat at 10 micrograms/kg and has a sample throughput of about 25 samples per analyst per day.

Animals↗
PubMed1994

Capillary zone electrophoresis separation of kinins using a novel laser fluorescence detector.

1. Bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and des-Arg1-bradykinin were separated by capillary zone electrophoresis in an apparatus constructed in our laboratory which utilizes a novel N2 pulsed laser-induced fluorescence detector. 2. Detection limits of 1.2 fmol for fluorescamine-derivatized bradykinin and 90 attomol for O-phthaldialdehyde-derivatized bradykinin were achieved. 3. This powerful analytical tool is described and its successful application to the measurements of bradykinin after enzymatic release from blood is documented.

Animals↗
PubMedDate not supplied

Quantitation of sulfamethazine in pork tissue by thin-layer chromatography.

Our earlier method to detect and quantitate sulfamethazine (SMZ) in milk at the 10 ppb level was modified to quantitate SMZ in pork tissue. Sulfabromomethazine (SBZ) is added to the tissue as an internal standard. SMZ and SBZ are extracted from the tissue into water as the supernatant of a centrifuged, aqueous homogenate and are cleaned up and concentrated by a series of solid-phase extractions. The sulfonamide-containing eluate is then separated on a silica gel thin-layer chromatographic plate. SBZ and SMZ are derivatized with fluorescamine, and their fluorescence is quantitated with a scanning densitometer. The limit of detection was estimated at 0.25 ppb (signal-to-noise ratio, 3:1). The average accuracy over the analysis range (0.54-21.8 ppb [micrograms/kg]) was 95.6% (standard deviation = 29.4%, n = 54).

Animals↗
PubMedDate not supplied

Planar chromatography for the multiclass, multiresidue screening of chloramphenicol, nitrofuran, and sulfonamide residues in pork and beef.

A method is described for multiclass and multiresidue qualitative detection of chloramphenicol, nitrofuran, and sulfonamide residues in animal muscle. The drugs are extracted from 1 g tissue with 2 mL ethyl acetate and purified by silica solid-phase extraction. After elution of the cartridge, the collected solution is evaporated, and the residue is dissolved in methanol and chromatographed on a Si60 high-performance thin-layer chromatography plate. After evaporation of solvent, nitrofurans are visualized first by their specific UV photochemical reaction with pyridine. Then chloramphenicol is reduced to its amino derivative, and this derivative and the sulfonamides are visualized by long-wave UV after reaction with fluorescamine. Chloramphenicol, nitrofurans, and sulfonamides are detected at residue level of 10, 5, and 100 micrograms/kg, respectively, or less in pork and beef.

Animals↗
PubMedDate not supplied

Determination of sulfonamides in edible salmon tissue by liquid chromatography with postcolumn derivatization and fluorescence detection.

Fourteen sulfonamides-sulfanilamide, sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine, sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine, and sulfaquinoxoline-residues of which could be found in aquacultured species, were separated in < 25 min by reversed-phase (C18) liquid chromatography (LC) with gradient elution. Analytes were extracted from edible salmon tissue (muscle and adhering skin) with acetonitrile-2% aqueous acetic acid, isolated with 2 liquid-liquid partitionings, and derivatized with fluorescamine after eluting from the column. The derivatives were detected by fluorescence. Recoveries (n = 4) from coho salmon fortified with sulfonamides at 5, 10, and 20 ng/g tissue averaged 79.7 +/- 7.3, 84.6 +/- 7.7, and 88.2 +/- 7.1%, respectively. Limits of quantitation were 5 ng/g tissue, for sulfanilamide, sulfamethoxypyridazine, and sulfaquinoxoline and 1 ng/g tissue for the remaining sulfonamides.

Acetic Acid↗
PubMed1995

Formulation and antitumor efficacy of liposomal-caprylated-TNF-SAM2.

The tumor necrosis factor (TNF) mutant TNF-SAM2 has previously been shown to have a therapeutic profile superior to parental TNF. To initially evaluate the characteristics of liposomal formulations of TNF-SAM2, it was modified with the N-hydroxysuccinimide ester of caprylic acid to increase its hydrophobic binding to multilamellar and small unilamellar vesicles (MLVs and SUVs). Native PAGE and fluorescamine analysis of acetylated parental TNF and TNF-SAM2 indicated that these proteins both displayed trimeric structures based on crosslinking/SDS-PAGE analysis and behaved similarly with respect to reactivity of their amino functions. Limited N-terminal sequencing analysis of partially acetylated (approx 3 acetyl groups per trimer) TNF-SAM2 indicated that the N-terminal Val was not modified; this was also concluded based on HPLC/mass spectrometric (LC-MS) analysis of Glu C digests. LC-MS analysis of tryptic digests of the acetylated TNF-SAM2 indicated that Lys-98 was unreactive. Molecular ions corresponding to acetylated Lys-containing peptides for all five other Lys residues could be detected; none appeared hyperreactive, but Lys-11 appeared hyporeactive. MLVs composed of DMPC/DMPG (7:3) and SUVs composed of DPPC/DSPC (1:1) displayed high capacity for binding to caprylated TNF-SAM2. These formulations of caprylated TNF-SAM2 displayed tumor necrotizing and growth-inhibitory activity in a syngeneic tumor model, and may be candidates for clinical development.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily.

By using two molecular probes, we demonstrate that only 4% of total phosphatidylethanolamine (PtdEtn) in the plasma membrane (PM) of a human pathogenic yeast, Candida albicans, is present in its external half. Evidence is presented to show that the availability of PtdEtn could be related to the expression of a multidrug transporter CDR1 of C. albicans, and the process is energy-dependent. A homozygous CDR1 disruptant strain of C. albicans shows almost 23% reduction in the external labelling of PtdEtn. This report shows that, similar to human MDRs, yeast multidrug transporter could also be involved in aminophospholipid translocation.

ATP-Binding Cassette Transporters↗
PubMed1995

Efficient analysis of cytochrome P4501A catalytic activity, porphyrins, and total proteins in chicken embryo hepatocyte cultures with a fluorescence plate reader.

An efficient method for measuring polychlorinated biphenyl-mediated induction of cytochrome P4501A, using the ethoxyresorufin-O-deethylase (EROD) assay, and total porphyrins in chicken embryo hepatocyte cultures is described. Hepatocytes were cultured in 48-well plates, assays were carried out within the wells, and concentrations of the product of the EROD reaction (resorufin), porphyrins, and total proteins were measured in the same wells with a fluorescence plate reader. The method differs from previous methods developed in this laboratory in that three fluorophors were measured in the same well rather than two.

Animals↗
PubMed1997

A continuous method for measuring calpain activity.

A simplified methodology for assaying the caseinolysis by calpains was developed. This methodology, including the incubation of calpain with casein and direct measurement of the absorbance at 500 nm, is based on the turbidity of the reaction mixture caused by the aggregation of hydrolysates during the reaction. Unlike the typical caseinolysis assay, this novel assay does not need to separate the substrate from hydrolysates and can be continuously monitored in visible wavelength range. The activity of calpain is expressed by the maximum reaction velocity (delta A500/min) at 25 degrees C).

Animals↗
PubMed1996

Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-O-deethylase induction in H4IIE cells: implications for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons.

A recombinant H4IIE rat hepatoma cell line (H4L1.1c4, H4IIE-luc), containing a luciferase reporter gene under control of dioxin-responsive enhancers, was examined for responsiveness to several polyhalogenated aromatic hydrocarbons (PHAHs). The recombinant cell system was compared with the widely used wild-type cell line (H4IIE-wt), which expresses Ah receptor-mediated cytochrome P450 1A induction. We also report an improved and down-scaled method for the H4IIE-wt bioassay which allows for the rapid screening of environmental samples for Ah-active PhAHs. This method employs 96-well plates, a plate-reading spectrofluorometer, and a fluorescence-based protein assay that enables the simultaneous measurement of resorufin and protein. Both cell lines demonstrated a dose-dependent increase in Ah receptor-mediated response upon exposure to a number of known Ah receptor agonists, including Halowax 1014. H4IIE-luc cells were 3-fold more sensitive than H4IIE-wt cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The detection limit and ED50 for EROD induction by TCDD were 0.6 and 4.9 fmol/well (2,4 and 20 pM), respectively; for luciferase induction they were 0.2 and 1.4 fmol/well (0.8 and 5.6 pM). The detection limit for EROD induction in H4IIE-wt cells was a 50-fold improvement over that reported previously (Tillitt et al., Environ. Sci. Technol. 25, 87-92, 1991) and comparable to that of a chicken embryo primary hepatocyte bioassay (Kennedy et al., Anal. Biochem. 211, 102-112, 1993). The tested PHAHs exhibited a similar structure-activity relationship in H4IIE-luc as in H4IIE-wt cells. Binary mixtures of TCDD, PCB-126, and PCB-77 showed no departure from additivity in their combined responses when tested in H4IIE-wt cells. PCB-153 at the highest tested dose of 14 nmol/well (56 microM) significantly reduced the potency of TCDD and PCB-126 without affecting their efficacy in both H4IIE-wt and H4IIE-luc cells. These findings support the use of H4IIE-luc cells as an alternative bioanalytical tool to the wild-type cells for the detection of Ah agonists in environmental samples.

Animals↗