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At least 577 records · Page 32Linked to original sources
PubMed1992

ATP-stimulated protease activity in brown fat mitochondria: response to a 24-h fast in mice.

Brown fat mitochondria have [3H]casein-hydrolyzing activity at pH 8.0 associated with both membrane and soluble fractions. An ATP-stimulated proteolytic activity inhibited by vanadate and N-ethylmaleimide was found in the soluble fraction. Membrane-associated proteolytic activity was inhibited by phenylmethylsulfonyl fluoride and trypsin inhibitor, suggesting that it is a serine protease. A 24-h fast in mice caused a significant loss of mitochondrial proteins from the tissue, but had no effect on protease activity of isolated mitochondria with or without ATP. The ATP-stimulated release of amino acids or peptides from isolated mitochondria, as measured with fluorescamine, was not influenced by food deprivation. Thus, brown fat mitochondria possess an ATP-stimulated proteolytic pathway that does not appear to be involved in the bulk removal of mitochondrial proteins from brown fat of fasting mice.

Adenosine Triphosphate↗
PubMed1989

Changes in the regional brain histamine and histidine levels in postmortem brains of Alzheimer patients.

In postmortem brains of Alzheimer patients, statistically significant decreases in histamine levels were observed in the frontal (45%), temporal (20%), and occipital cortices (38%) and in the caudate nucleus (25%). Histidine levels were decreased in the frontal (15%), temporal (21%), and occipital cortices (30%) and in the caudate nucleus (25%); the decrease was statistically significant in the last two brain regions. Histamine was determined by the double isotope technique, and histidine was determined fluorometrically by a fluorescamine method. The data indicate that brain histamine regulation is altered in Alzheimer's disease.

Aged↗
PubMed2002

Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation.

To determine whether homocysteine (Hcy)-mediated activation of endocardial endothelial (EE) cells is ameliorated by peroxisome proliferator-activated receptor (PPAR), we isolated EE cells from mouse endocardium. Matrix metalloproteinase (MMP) activity and intercellular adhesion molecule (ICAM)-1 in EE cells were measured in the presence and absence of Hcy, and ciprofibrate (CF; PPAR-alpha agonist) or 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2); PPAR-gamma agonist) by zymography and Western blot analyses, respectively. Results suggest that Hcy-mediated MMP activation and ICAM-1 expression are ameliorated by CF and PGJ(2). To test the hypothesis that Hcy competes with other ligands for binding to PPARalpha and -gamma, we prepared cardiac nuclear extracts. Extracts were loaded onto an Hcy-cellulose affinity column. Bound proteins were eluted with CF and PGJ(2). To determine conformational changes in PPAR upon binding to Hcy, we measured PPAR fluorescence at 334 nm. Dose-dependent increase in PPAR fluorescence demonstrated a primary binding affinity of 0.32 +/- 0.06 microM. There was dose-dependent quenching of PPAR fluorescence by fluorescamine-homocysteine (F-Hcy). PPAR-alpha fluorescence quenching was abrogated by the addition of CF but not by PGJ(2). PPAR-gamma fluorescence quenching was abrogated by the addition of PGJ(2) but not by CF. These results suggest that Hcy competes with CF and PGJ(2) for binding to PPAR-alpha and -gamma, respectively, indicating a role of PPAR in amelioration of Hcy-mediated EE dysfunction.

Animals↗
PubMed1998

Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells.

Dynamic changes in the reduction-oxidation (redox) state of the tissue lead to the pathophysiological condition. Reduced homocysteine causes dysfunctions in endothelium. The proliferation of smooth muscle cells may lead to occlusive vascular disease, ischemia, and heart failure, but whether fibrosis and hypertension are a consequence of smooth muscle proliferation is unclear. Redox changes during hyper-homocyst(e)inemia may be one of the causes of premature atherosclerotic heart disease. To examine the effect of homocystine on human vascular smooth muscle cells (HVSMC), we isolated HVSMC from idiopathic dilated cardiomyopathic hearts. Coronaries in these hearts were apparently normal. HVSMC numbers in culture were measured by hemocytometer in the presence and absence of homocystine. Results show that homocystine induced cellular proliferation. This proliferation was reversed by the addition of the antioxidant N-acetylcysteine (NAC). Homocystine induces collagen expression in a dose- and time-dependent manner, as measured by Northern blot (mRNA) analysis. The 50% inhibitory concentration of 5 microM for collagen was estimated. The induction of collagen was reversed by the addition of NAC and reduced glutathione. To localize the receptor for homocystine on HVSMC, we synthesized fluorescamine-labeled homocystine conjugate. Incubation of labeled homocystine with HVSMC demonstrated membrane and cytosol localization of homocystine binding. The receptor-ligand binding was disrupted by NAC. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography, we observed a 40- to 25-kDa homocystine redox receptor in HVSMC. Our results suggested that the redox homocysteine induces HVSMC proliferation by binding to the redox receptor and may exacerbate atherosclerotic lesion formation by inducing collagen expression.

Acetylcysteine↗
PubMed1993

Transbilayer mobility and distribution of red cell phospholipids during storage.

We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.

Adenosine Triphosphate↗
PubMed1992

A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B1 and B2.

A thin layer chromatography (TLC) method was developed for the detection of fumonisin B1 and B2 in corn and corn-based feedstuffs. Finely ground samples were extracted with acetonitrile:water (1:1), filtered, and applied to C18 cleanup columns. The columns were washed with 1% aqueous KCl followed by acetonitrile: 1% aqueous KCl (1:9), and the fumonisins were eluted with acetonitrile:water (7:3). The eluants were concentrated and spotted on reverse-phase C18 TLC plates along with fumonisin B1 and B2 standards, and the plates were developed in methanol: 4% aqueous KCl (3:2). The fumonisins were visualized by spraying the TLC plates successively with 0.1 M sodium borate buffer, fluorescamine, and 0.01 M boric acid. The plates were then dried and examined under longwave ultraviolet light. Fumonisin B1 and B2 appeared as bright yellowish-green fluorescent bands at Rfs of 0.5 and 0.1, respectively. The detection limit for the fumonisins on the TLC plate was 0.1 ppm in corn. Recoveries from spiked samples averaged greater than 80%. The identification of the fumonisins was confirmed by hydrolyzing the parent compounds of B1 and B2 to their respective C22 amino-alcohols and reexamining by TLC with the same visualizing reagents. This procedure was used to survey 193 corn samples collected from University of Missouri test plots in 1990 for fumonisin B1. Fumonisin B1 was detected in 15% of the corn samples.

Animal Feed↗
PubMed1976

Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus-transformed cells and Hydra attenuata.

The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.

Autoanalysis↗
PubMed1975

Deficiency of arginine esterase in cystic fibrosis of the pancreas: demonstration of the proteolytic nature of the activity.

Proteolytic activity, defined as the hydrolysis of peptide bonds involving the carboxyl group of L-arginine, in plasma of patients with cystic fibrosis, heterozygotes, and control subjects has been assayed using a fluorometric method with protamine as the substrate and fluorescamine as the reagent. The mean total proteolytic activity in plasma of patients with cystic fibrosis was approximately one-half the mean total activity in control subjects and heterozygotes. The mean proteolytic activity inhibited by soybean trypsin inhibitor in plasma of patients with cystic fibrosis was approximately one-third that of control subjects and heterozygotes. The relationship of arginine esterase activity to proteolytic activity was investigated. The pH optimum and action of reversible and irreversible inhibitors were similar for both activites, suggesting that the arginine esterase activity and proteolytic activity represent similar catalytic entities. These findings are consistent with our hypothesis that the basic defect in cystic fibrosis may reside in the deficieny of a proteolytic enzyme which results in the accumulation of the various cationic macromolecular "factors" described by other investigators in serum of patients with cystic fibrosis. The demonstration of a deficiency of proteolytic activity as assayed by the hydrolysis of protamine, a cationic polypeptide, could explain the presence of ciliotoxic cationic protein or polypeptide factors in serum of patients with cystic fibrosis and may, in some unknown manner, be related to the clinical manifestations of the disease.

Adolescent↗
PubMed1990

Determination of triethylenetetramine in plasma of patients by high-performance liquid chromatography.

A sensitive and simple fluorometric method for the determination of N,N'-bis(2-aminoethyl)-1,2-ethanediamine dihydrochloride (triethylenetetramine) in human plasma by high-performance liquid chromatography is described. Free triethylenetetramine (TETA) obtained by passing the TETA-copper chelate compound through a solid-phase cation exchange resin was converted to its fluorescamine derivative in the presence of ethylenediaminetetraacetic acid to mask the interfering metal ions in the reaction solution, and the derivatives were separated on a nitrile high-performance liquid chromatograph column (Nucleosil 5-CN) using isocratic elution. The plasma levels of TETA were measured in eight patients receiving treatment for excess copper. Absorption rates of TETA were relatively slow and the peak levels were significantly different among patients. The bioavailability of TETA in the rat was also examined and the ratio of intestinal absorption was extremely low.

Animals↗
PubMed1977

Microassay methods for taurine and cysteine sulfinate decarboxylase activity.

Microassay methods for taurine (2-aminoethanesulfonic acid) and cysteine sulfinate decarboxylase (CSD: EC 4.1.1.29) have been developed. Cation exchange resin (Dowex 50W x 8, 200--400 mesh) was used for the separation of taurine from other fluorescamine reactive substances. The "taurine fraction" in this column chromatographic procedure was collected and fluorescence development was carried out. The maximal sensitivity obtained for taurine was 25 pmoles. The specificity, reproducibility, sensitivity and recovery for taurine obtained by this method were satisfactory enough to be used for biological applications. The activity of CSD was determined by measuring the formation of 14CO2 from DL-[1-14C]-cysteine sulfinic acid using a specially designed micro-vessel. The activity in cerebral tissues containing 5 microgram of protein was accurately detected by this microradiometrical method. Using above microassay methods for taurine and CSD activity, it was found that in the rat spinal cord, taurine is distributed rather evenly, whereas the distribution of CSD activities is uneven and the highest activity is localized at the dorsal part of the dorsal horn.

Animals↗
PubMed1977

The anatomy of cooperative binding between protamines and DNA.

DNA was titrated with protamine under equilibrium conditions while binding was simultaneously followed by electron microscopy and the fluorescamine technique. Positive cooperativity concluded from the binding assays was shown to be correlated with an aligned association of DNA fibres. Subsequent structural modifications, dependent upon the protamine/DNA ratio, suggests a mechanism for DNA compaction by protamines. In the framework of this model, protamine phosphorylation appears to promote formation of DNA interstrand links yielding a very pronounced filigree structure.

Clupeine↗
PubMed2000

Eimeria acervulina infection elevates plasma and muscle 3-methylhistidine levels in chickens.

To assess muscle breakdown during avian coccidiosis, the level of the nonmetabolizable amino acid 3-methylhistidine (3MH) was determined in muscle, plasma and excreta from chickens infected with Eimeria acervulina. The changes in 3MH levels during infection were assessed at 1-29 days postinoculation (DPI) in animals given 5 x 10(5) oocysts per bird. The effect of levels of parasitism were evaluated at 8 DPI in birds receiving 5 x 10(3), 5 x 10(4), 5 x 10(5) or 1 x 10(6) oocysts each. The 3MH levels of plasma, muscle, and excreta samples were determined by high-pressure liquid chromatography after derivatization with fluorescamine. Weight gains, breast muscle weight, eviscerated weight, plasma carotenoid levels, dry weight of muscle, and gross lesion scores were also determined. Infected birds had significantly elevated plasma and muscle 3MH at 4 and 8 DPI following a single dose of E. acervulina. The increase in 3MH levels had an inverse relationship with the time course of weight gain and plasma carotenoid levels. Plasma and muscle 3MH levels returned to control values by 15 DPI and remained unchanged from control values through the remainder of the experiment (29 DPI). Breast weight was decreased in infected birds, but the ratio of breast weight to eviscerated body weight was unchanged. Excretion of 3MH decreased relative to controls at 4 and 8 DPI and returned to control levels on 15 DPI. The plasma and muscle levels of 3MH were related to severity of infection; however, levels of excreted 3MH were not. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of E. acervulina infection. The underlying causes for this muscle breakdown was unclear but could involve a physiological response to anorexia and decreased food intake during the acute phase of infection. Levels of excreted 3MH did not increase during infection and this may be the result of decreased excreta output during infection. Plasma and muscle levels of 3MH were correlated with severity of E. acervulina infections but may not be as sensitive an indicator of infection as plasma carotenoid levels or other physiological parameters.

Animals↗
PubMed1988

Nitrogen-to-protein conversion factors in animal feed and fecal samples.

Nitrogen-to-protein conversion factors were calculated for diets and fecal samples from three animal species fed low- or high-fiber diets. The conversion factors based on protein contents determined by amino acid analyses were calculated from total N (Kjeldahl analyses) and recovered amino acid plus NH3-N (amino acid analyses). Conversion factors based on protein contents determined by a fluorescamine assay for amino acids were also calculated from total Kjeldahl-N. The conversion factors based on Kjeldahl-N averaged 5.3 +/- .7 for the diets and 4.0 +/- .5 for the feces in the six animal and diet comparisons when calculated from anhydrous amino acid formula weights. The greatest deviations from the traditional 6.25 conversion factor occurred in the fecal samples of ruminant animals fed a corn-alfalfa meal diet at a maintenance level of intake. In contrast, conversion factors based on amino acid plus NH3-N were quite stable. These factors averaged 5.7 +/- .1 for feeds and 5.5 +/- .1 for feces when calculated from anhydrous amino acid residue weights.

Amino Acids↗
PubMed1979

The turnover of myelin proteins in adult rat brain.

The turnover of proteins of myelin of brain was followed after an intracerebral injections of [1-14C] leucine into adult rats and compared with the turnover of proteins of total brain homogenate, microsomal and supernatant fractions. The myelin proteins were separated into three major fractions, viz., the Wolfgram, proteolipid and basic proteins as fluorescamine derivatives by a new preparative sodium-dodecylsulfate-polyacrylamide slab gel electrophoresis method. The bands were visualized, without staining, by their fluoresence under UV light. The maximal incorporation into myelin proteins was about 11% of that in the microsomal proteins. The maximum amoount ofnto myelin proteins was about 11% of that in the microsomal proteins. The incorporation of the label in myelin proteins reached a moximum about six hr after the injection, while the microsomal and supernatant and myelin proteins declined at multiphasic rates with half-lives varying from three hours to several days. Individual myelin proteins also appeared to turn over at multiphasic rates, with half-lives of 5, 20 and 90-100 days for the Wolfgram proteins, 7 and 100-120 days for the proteolipid proteins and 5 and 80-110 days for the basic proteins.

Animals↗
PubMed1984

A rapid sensitive assay for the quantitation of elastin.

A rapid, sensitive and direct assay was developed for the quantitation of elastin using reverse phase high performance liquid chromatography. The method is based on the separation of the alkaline hydrolysis-resistant dipeptide valyl-proline, a dipeptide found in high concentrations in elastin. The methodology is applicable to unlabelled elastin samples in the range of 10-100 micrograms utilizing fluorescamine detection and to hot alkali insoluble as well as soluble forms of elastin. Increased sensitivity was achieved by the use of in vitro radiolabelling of elastin with [3H]proline. This was useful for following synthesis of elastin in cultured cells. Since this technique is not dependent on detection of the elastin-specific crosslink groups, desmosine and isodesmosine, precursor forms of elastin can be measured as well as other forms of elastin deficient in cross-linking. The same chromatographic procedure however, is able to detect these desmosine and isodesmosine crosslinks. Therefore, in cell cultures under the proper radiolabelling conditions, biosynthesis and cross-linking of elastin can be followed simultaneously in a single chromatographic procedure.

Animals↗
PubMed1978

Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.

Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluorometrically: propidium iodide (PI) with fluoresceinisothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPII) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitrobenzodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combination revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest. With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.

Animals↗
PubMed1990

Fluorescence detection of carbon-centered radicals in aqueous solution.

A simple, highly sensitive method for the simultaneous determination of arrays of carbon-centered radicals in aqueous systems is described. Radicals are efficiently trapped by an amino-nitroxide to form stable products which are then reacted with fluorescamine to produce highly fluorescent adducts. The adducts are easily separated by reversed-phase high performance liquid chromatography. The detection limit for individual radical adducts (0.5 to 2 nM) is two to three orders of magnitude lower than those of current methods employing electron paramagnetic resonance detection. Results on the photolysis of ketones and alpha-keto acids demonstrate the potential of this technique. This approach should be widely applicable to the study of radical processes in biological and chemical systems.

Chemical Phenomena↗
PubMed1980

Long-term function of NCGT vascular conduits in a multicenter trial: evaluation of physical chemical parameters.

Our experience with the NCGT graft has now encompassed 12 years in experimental animals and 4.5 years clinical experience with 134 grafts in man. It has previously been suggested that vessel wall structure, interface-charge, electric potential, and polarity of the blood intimal interface appear critical in the prevention of intravascular thrombosis in all vascular prosthetic bypass grafts. This concept has now been confirmed using ferritin, colloidal iron and fluorescamine intimal labelling. These provide a quantitative special characterization of the surface charges of several grafts, including the NCGT graft. Study indicates that each step in the production of the NCGT graft results in a cumulative increase in the structured negative charge of the vascular interface. The more dense the structured negative charge of the prosthesis, the more resistant is the graft to short and long-term thrombosis in experimental animals and man. The experience has been confirmed with a comparative analysis of implantation results in 105 patients up to 4.5 years with 65 to 70% patency rate over that time interval. Statistical analysis of parameters confirm again that the polarity and structure of the vascular interface is important in the effective function and patency rate of the grafts.

Animals↗