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PubMed2003

A bifunctional alkylating nitrogen mustard agent that utilizes barbituric acid as carrier drug with the potential for crossing the brain-blood barrier.

Barbituric acid is the parent compound of a large family of hypnotic barbiturates. A nitrogen mustard (N-mustard) group (-CH2CH2N[CH2CH2Cl]2) was placed onto the two nitrogen atoms at positions 1 and 3 of the pyrimidine ring. This N-mustard agent is a solid at 25 degrees C, stable at -10 degrees C for >10 weeks, and soluble in aqueous solvent at 37 degrees C and 25 degrees C. The partition coefficients miLog P and CLog P were calculated to be -0.93 and -1.441 for barbituric acid. The miLog P and CLog P for the N-mustard agent were 1.82 and 2.707, respectively. The N-mustard substituents significantly increased solubility in lipid by-layers. The N-mustard agent alkylated a nucleophilic primary amine (p-chloroaniline) at physiological conditions of pH 7.4 and 37 degrees C. Aliquots of reaction mixtures were withdrawn at known time periods to react with fluorescamine for determination of unreacted p-chloroaniline and calculation of rate constants. The alkylation of the primary amine was second order with rate = k2[Nu]2, (Nu is nucleophile) and rate constant k2 = 0.01358 L/(mole.min). The molecular dipole of barbituric acid and the N-mustard agent was calculated by SPARTAN software (wavefunction, Irvine, CA) to be 0.681 and 2.153 Debye, respectively. The brain/blood partition coefficient (Log BB) of the N-mustard agent was -0.399. Values of molecular polar surface area (TPSA) for barbituric acid and the N-mustard agent was 75.27 and 64.17, respectively. TPSA values indicate an expected intestinal absorbance to be 79% and 90%, respectively. The N-mustard agent showed zero violations of the Rule of 5, indicating good bioavailability.

Barbiturates↗
PubMed1977

Clearance of sulfamethoxazole in eggs and tissues of chickens.

Clearance of sulfamethoxazole (SMX, or sulfisomezole) in eggs and tissues of chicken after withdrawal of the drug which was medicated feed additively (at 0.2 or 0.4%) or administered intramuscularly (100 mg./kg., twice daily) for 5 successive days was determined using a fluorescamine reagent. Clearance patterns of SMX were found to be different between albumen and yolk. SMX level in albumen diminished linearly to below 0.1 p.p.m. (sensitivity level) by 5 days after the cessation of medication. On the other hand, SMX level in yolk remained at a plateau until 4 days, and thereafter decreased linearly to below 0.1 p.p.m. by 10 days. SMX levels in most tissues diminished below 0.1 p.p.m. by 3 days and those in kidney and skin by 4 days. Clearance patterns in tissue were quite similar to those in egg albumen. Proportion of acetylated-SMX to total-one in both albumen and yolk tended to increase up to 10% with the lapse of time after drug withdrawal. Acetylation was high in liver (36.5-43.6%), spleen skin and fat, but low in breast muscle and kidney (3.7-11%) in the groups of feed addition; while acetylation was low in liver of the group of intramuscular injection.

Administration, Oral↗
PubMed1988

Identification of fluorescent glycopeptide derivatives by two consecutive high pressure liquid chromatographic procedures.

Reversed phase high pressure liquid chromatography (HPLC) was used to separate individual components of the complex glycopeptide antibiotic teicoplanin in microgram quantities with gradient elution. Each of eight different fractions was then subjected to a specific and highly sensitive HPLC method, which has been developed for the determination of teicoplanin concentrations in biological specimens. This analytical procedure includes precolumn derivatization with fluorescamine and isocratic elution. The fluorescent teicoplanin derivatives were identified by comparing their retention times in both HPLC procedures. Derivatization resulted in increased hydrophobicity and improved chromatographic separation, but the order of elution of the different compounds was not changed. The antimicrobial activity of the individual underivatized fractions correlated with their respective contents of total teicoplanin A2, whereas the pseudo-aglycone A3 appeared less active. Similar techniques have the potential to be applied to other complex glycopeptide antibiotics.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Characteristics of bacterioplankton in the Loess-containing Lake Khanka].

Some characteristics of bacterioplankton--generation time, daily (P) and specific (P/B) bacterioplankton production, and bacterial metabolic coefficient K2--in the loess-containing Lake Khanka were determined using five modifications of the bacterial-count procedure with the fluorescent dyes fluorescamin and erythrosin. Experiments showed that the organomineral complex (OMC) in this lake is broken down by chemoorganoheterotrophic bacteria. The increase in the loess content of the lake water intensified bacterial growth and the cycles of potassium, silicon, and other biogenic elements. The addition of starch to a loess suspension activated the breakdown of OMC due to the adsorption of starch on the OMC/water interface and stimulation of the metabolism of attached bacteria.

Animals↗
PubMed1979

[Electrophoretic profiles of proteins and glycoproteins of chick embryo fibroblasts during development].

The variations of proteins and glycoproteins of Chick embryo fibroblasts are studied during development. This investigation is carried out using polyacrylamide disc gel electrophoresis in SDS. Two glycoproteins of high apparent molecular weight (250,000 and 200,000) undergo quantitative modification: they increase from the 8th to 12th day of development and then remain unchanged to the 16th day. They are cell surface components as suggested by fluorescamine labelling and trypsin sensitivity. The results are discussed in terms of relationship between tumor- and embryo cells.

Animals↗
PubMed1975

Fluorometric determination of nitrite in cured meats.

An indirect fluorometric method for determining sodium nitrite in meat products is presented. The extracted sodium nitrite is consumed in a diazotization reaction with a measured excess of sulfanilic acid. Fluorescamine, which acts selectively with primary amines such as sulfanilic acid, is a fluorogenic reagent for the excess amine. The amine consumed, calculated by difference from the total originally present, is directly related to the sodium nitrite content of the sample. Interferences from amino acids and soluble proteins in the meat extract are eliminated by judicious use of a secondary peak in the fluorescence spectra (436 nm excitation, 495 nm fluorescence) combined with measurement at low pH (3.30). The recoveries of sodium nitrite ranged from 83.2 to 99.6% with an average of 93.4 and a standard deviation of +/- 5.28% for 11 determinations.

Food Additives↗
PubMed1975

The release of glutamic acid from isolated brain tissues.

The glutamic acid and other primary amines released spontaneously and by electrical pulses from rat brain caudate and cortex were measured by the fluorescamine method. Electrical pulses evoked a transient release of glutamic acid which was greater from caudate tissue. Both morphine and reserpine, in vivo, inhibited the spontaneous glutamic acid release but not the loss of glutamic acid due to pulses.

Animals↗
PubMed1992

[In vitro effects of low molecular weight dextran on the surface properties of LDL].

The relative number of free amino groups on the surface of low density lipoproteins (LDL) was determined after the incubation with low molecular weight dextran (MW 40,000) in several concentrations by two different assays (fluorescamine, TNBS). A decrease of the detectable free amino groups of 40% was shown by both assays. After the incubation of lysine with dextran these changes could not be observed. These results are explained with the aggregation of LDL particles influenced by dextran.

Dextrans↗
PubMed1992

Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.

Animals↗
PubMedDate not supplied

Determination of sulfonamide residues in the tissues of food animals using automated precolumn derivatization and liquid chromatography with fluorescence detection.

A liquid chromatographic method for the determination of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfadoxine, sulfaethoxypyridazine, sulfamethazine, sulfaquinoxaline, and sulfathiazole residues in the muscle, liver, and kidney of food animals using sulfapyridine as internal standard is reported. Tissues are extracted using a modified version of AOAC Official Method 983.31 (Sulfonamide Residues in Animal Tissues). The sample extract is reconstituted in pH 3.0 buffer-acetonitrile (60 + 40) and filtered into an autosampler vial. Using a programmable autosampler of a liquid chromatograph, a portion of the sample is derivatized precolumn with fluorescamine. The sulfonamide derivatives are separated by liquid chromatography using a C18 column with a mobile phase of 0.02M phosphoric acid-acetonitrile (60.5 + 39.5) and detected by fluorescence (excitation, 405 nm; emission, 495 nm). The method was applied to swine and cattle muscle, liver, and kidney; sheep and horse muscle and kidney; and chicken muscle and liver. The mean values for samples fortified with sulfonamides at levels between 0.05 and 0.2 microg/g agreed within 96-99% of spiked levels, with coefficients of variation ranging from 4-10%. The limit of detection (LOD) for all sulfonamides was 0.01 microg/g, with the exception of sulfaquinoxaline, for which the LOD was 0.015 microg/g.

Animals↗
PubMed1991

Fluorescence studies on aged and young erythrocyte populations.

Structural changes in red blood cell (RBC) membrane are investigated by fluorescence techniques. Results obtained with three probes (DPH, 3-PM and fluorescamine) indicate a significant increase in membrane rigidity associated with aging of RBCs. Discrepancies between our observations and published data could arise from utilization of experimental conditions closer to physiological conditions in our study. Use of intact RBCs continuously manipulated in a 37 degrees C environment could represent experimental conditions favourable to the identification of rheologic membrane changes in senescent RBCs.

Erythrocyte Aging↗
PubMed1990

[Interaction of tobramycin and its derivatives with mitochondria in vitro].

Tobramycin modified with fluorescamin yields fluorescent derivatives with a gradually changed structure and charge composition. Interactions of these drugs with mitochondria were investigated on the basis of the measurement of their inhibitory effect on the respiration (consumption of O2) of the isolated mitochondria. The results were compared with the effects of the original antibiotic agent. The use of the method of fluorescence polarization made it possible to determine the character of interactions. The procedure is a model experiment with possible wider use.

Animals↗
PubMed1990

[Cytochemical analysis of polyamines in Ehrlich ascitic carcinoma cells during different phases of mitotic cycle under the effects of difluoromethylornithine].

Mice bearing Ehrlich carcinoma cells received one 80 mg/kg dose of DFMO. After 20 hours the cells were extracted from the peritoneal cavity and separated by means of sedimentation of 1 g in saccharose gradient into 9 fractions depending on the mitotic cycle phase. The content of polyamines in the cells of each fraction was detected by the fluorescent method using fluorescamine. It was shown that the tumor cells during G1 phase mitotic cycle (13-15% cells blocked) and especially cells in the 1st half of S-phase (19-23% cells blocked) were most sensitive to inhibitory effect of DFMO.

Animals↗
PubMed1985

[Electrophoretic purification of biologically active structural proteins of the simian adenovirus SA7 in the presence of sodium dodecyl sulfate].

The modification of disc electrophoresis technique in polyacrylamide gel with sodium dodecylsulphate (SDS) has been elaborated for synchronous isolation of some structural proteins in biologically active form and in preparative quantities from adenoviruses. Virions of SA7 adenovirus were mildly dissociated in SDS solution at 20 degrees C and structural proteins were stained by fluorescamin. After separation the zones of proteins corresponding to the native capsomeres of hexon and protein IV as well as the zones of inner proteins V and VII have been identified as fluorescent at UV-irradiation, excised and extracted by SDS solution. After the removal of SDS by protein precipitation in acetone the preparations of hexon and IV reveal the quaternary structure of native capsomers and full spectrum of antigenic and immunogenic activities of native proteins. Preparations of inner proteins V and VII possess activity in condensing adenoviral DNA. The technique is usable for preparative purification of inner polypeptide VI SA7, as well as capsomers and inner proteins of other adenoviruses.

Adenoviridae↗
PubMed1989

Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the guanine nucleotide exchange factor as a GTP-binding protein.

Polypeptide chain initiation in mammalian systems is regulated at the level of the guanine nucleotide exchange factor (GEF). This multisubunit protein catalyzes the exchange of GDP bound to eukaryotic initiation factor 2 (eIF-2) for GTP. Although various models have been proposed for its mode of action, the exact sequence of events involved in nucleotide exchange is still uncertain. We have studied this reaction by three different experimental techniques: (a) membrane filtration assays to measure the release of [3H]GDP from the eIF-2.[3H]GDP binary complex, (b) changes in the steady-state polarization of fluorescamine-GDP during the nucleotide exchange reaction, and (c) sucrose gradient analysis of the total reaction. The results obtained do not support the reaction as written: eIF-2.GDP + GEF in equilibrium eIF-2.GEF + GDP. The addition of GEF alone does not result in the displacement of eIF-2-bound GDP. The release of bound GDP is dependent on the presence of both GTP and GEF, and this argues against the possibility of a substituted enzyme (ping-pong) mechanism for the guanine nucleotide exchange reaction. An important finding of the present study is the observation that GTP binds to GEF. The Kd value of 4 microM for GTP was estimated (a) by the extent of quenching of tryptophan fluorescence of GEF in the presence of GTP and (b) by the binding of [3H]GTP to GEF as measured on nitrocellulose membranes. The GEF-dependent release of eIF-2-bound GDP was studied at several constant concentrations of one substrate (GTP or eIF-2.GDP) while varying the second substrate concentration, and the results were then plotted according to the Lineweaver-Burk method. Taken together, the results of GTP and eIF-2.GDP binding to GEF and the pattern of the double-reciprocal plots strongly suggest that the guanine nucleotide exchange reaction follows a sequential mechanism.

Animals↗
PubMed1986

Localization and possible function of polyamines in protein and peptide secreting cells.

Three different cytochemical methods, the formaldehyde-fluorescamine and the ortho-phtalaldehyde fluorescence cytochemical methods as well as immunocytochemistry have been developed for localising the polyamines spermidine and spermine in tissue. All three methods produce identical results and show polyamines to occur inter alia in high concentrations in certain protein- and peptide-secreting cells. Many of these cells also show the capacity to metabolise monoamines and belong to the amine content or amine precursor uptake and decarboxylation (APUD) series. In cell types where fluorescence microscopical resolution allows, most polyamines appear to be localised to secretory granules. Moreover, studies on isolated pancreatic islets reveal active and glucose-dependent polyamine biosynthesis to occur in insulin cells. Possible function of polyamines in secretory granules are discussed in the light of the above findings.

APUD Cells↗
PubMed1987

The energy utilized in protein breakdown by the ATP-dependent protease (La) from Escherichia coli.

A crucial enzyme in the pathway for protein degradation in Escherichia coli is protease La, an ATP-hydrolyzing protease encoded by the lon gene. This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues. To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved. The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4 fold in rates of hydrolysis of peptide bonds. Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups. These values appeared lower at 1 mM than at 10 mM Mg2+. The coupling between ATP and peptide bond hydrolysis appeared very tight. However, when the protease was assayed under suboptimal conditions (e.g. at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved. Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis.

ATP-Dependent Proteases↗
PubMedDate not supplied

Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins.

Calmodulin binds quantitatively to phenyl-Sepharose through its Ca2+-induced hydrophobic binding region. Troponin C and S-100 protein, as well as several other proteins present in rat tissues, also bind to phenyl-Sepharose in a Ca2+-dependent manner. While the Ca2+-dependent binding of calmodulin to phenyl-Sepharose is not altered appreciably by monovalent cations, they do appear to compete for Ca2+ binding to most of the other proteins, including S-100 protein, which exhibit Ca2+-induced interaction with phenyl-Sepharose. The selective elution of these proteins from the phenyl-Sepharose column can be achieved with a 0.5 M concentration of monovalent cations such as K+, Na+, and NH4+ in the presence of a low (100 microM) Ca2+ concentration. Calmodulin-binding proteins associated with calmodulin in crude cell extracts can prevent the interaction of calmodulin with the phenyl-Sepharose, resulting in low recoveries of calmodulin from these tissues. The majority of these interfering proteins are heat labile so that heat treatment (boiling) of the cell extract for a limited time (5 min) negates any binding of these proteins to calmodulin and allows the quantitative recovery of calmodulin by hydrophobic interaction chromatography. This procedure allows the rapid and quantitative recovery of highly purified calmodulin from both cytosolic and Triton X-100-solubilized particulate fractions prepared from various rat tissues. Calmodulin isolated in this manner can be accurately and reliably quantitated by direct protein determination with Coomassie brilliant blue dye or fluorescamine or by the cyclic nucleotide phosphodiesterase stimulation assay.

3',5'-Cyclic-AMP Phosphodiesterases↗