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At least 865 records · Page 48Linked to original sources
PubMed1983

Luminometric determination of FAD in subpicomole quantities.

Very small quantities of FAD were able to reactivate apo-D-amino acid oxidase. In the presence of D-alanine, luminol, horseradish peroxidase, and an excess of the apoenzyme, a quantitative luminometric determination of FAD was possible. The maximal photon emission measured in a bicarbonate buffer, pH 9.2, at 37 degrees C was proportional to the amount of FAD added. FMN, riboflavin, or 5-deazaflavin produced no chemiluminescence and had no inhibitory effect in the assay when added together with FAD. With this method, FAD could be quantitatively determined with high accuracy in perchloric acid extracts of animal tissue and bacteria.

Alanine↗
PubMed1976

Carcinoembryonic antigen: isolation of a sub-fraction with high specific activity.

Four sub-fractions of carcinoembryonic antigen have been obtained by chromatography of conventional CEA on Con A-Sepharose and their immunoreactive contents have been determined. Comparative studies have shown that a fraction eluted with 2% methyl glucoside (CEA 2B) had the highest activity, with a potency (60 u/mug) twice that of unfractionated CEA although appreciable activity was also found in the other fractions. The amino acid composition of CEA 2B is similar to that reported for conventional CEA but there is a lower content of neutral hexoses and a comparatively high content of N-acetylglucosamine. Experiments with a pool of sera from 11 patients with colonic cancer, which had been fractionated on Con A-Sepharose, have shown that nearly all the CEA activity was contained in a fraction eluted with 2% methyl glucoside. A convenient method of isolating CEA with high specific activity directly from perchloric acid extracts of tumour tissue is also described.

Acetylglucosamine↗
PubMed1991

In vivo 14N nuclear magnetic resonance spectroscopy of tumors: detection of ammonium and trimethylamine metabolites in the murine radiation induced fibrosarcoma 1.

The in vivo 14N nuclear magnetic resonance spectra of s.c. implanted murine radiation induced fibrosarcomas (RIF-1) display narrow resonances assignable to betaine and other trimethylamines and broad resonances due to amino acids and peptides. In 19 of the 41 tumors studied a distinct resonance from the ammonium ion is detectable. The accumulation of ammonium in the tumor to nuclear magnetic resonance detectable levels may result from glutaminolysis (a possible pathway for energy production in the tumor), from the degradation of peptides and proteins, or from the deamination of adenine nucleotides. Estimates of the tissue ammonium concentration were obtained from the in vivo tumor spectrum and the spectrum of the nonlabile trimethylamines in the perchloric acid extract. In the extract, the 14N resonances of betaine, carnitine, choline, phosphorylcholine, and glycerophosphorylcholine were resolved, and a relatively high level of tissue urea was observed. Spin-lattice relaxation times were obtained for the 14N nucleus of each of these metabolites in phosphate buffer.

Ammonia↗
PubMed1977

Androgen regulation of transferase II activity in the mouse kidney.

Castration of adult male mice reduced the ability of the transferase factors of the kidney to stimulate amino acid incorporation by polysomes in vitro. The administration of testosterone propionate (TP) to the mice for 11 days greatly increased the activity of the transferase fraction. The changes occurred only in transferase II. The induction of the increase in transferase activity was evident 24 h after the injection of TP and required a lag period of at least 12 h. The concentration of pH 5 enzymes (protein) was slightly decreased by castration and was restored by TP administration. The radioactivity in the hot perchloric acid extract of the protein after amino acid incorporation was increased but the activity of the pH 5 enzyme fraction on amino incorporation was not significantly changed by androgen administration.

Animals↗
PubMed1994

Brain N-acetyl-L-aspartic acid in Alzheimer's disease: a proton magnetic resonance spectroscopy study.

This study was performed in order to measure changes in brain N-acetyl-L-aspartic acid (NAA) in post-mortem brain tissue in Alzheimer's disease (AD) in comparison to normal control subjects using the technique of magnetic resonance spectroscopy. Brain tissue was obtained at autopsy and frozen until use, from seven patients diagnosed according to current research criteria for AD and 7 control subjects. Detailed clinical evaluations were available for all the dementia cases. Representative brain samples were obtained from three neocortical regions and a limbic region (parahippocampal gyrus) in white and grey matter. NAA was quantified on perchloric acid extracts using proton nuclear magnetic resonance (NMR) spectroscopy. Regional NAA did not vary significantly with age. In AD, reductions were present in the grey matter of the neocortex but not in the white matter. Within the parahippocampal gyrus there were reductions in both tissue types; only cortical levels correlated with clinical scales of dementia severity. A pattern of increasing correlation was observed between dementia severity as measured by the mini mental state examination during life and NAA levels from brain areas of increasing pathological predilection in AD. These post-mortem studies show reductions in brain NAA in AD which correlate with dementia severity during life and which support the use of future in vivo NAA spectroscopic images in the evaluation of AD patients.

Aged↗
PubMed1997

A simple mathematical model and practical approach for evaluating citric acid cycle fluxes in perfused rat hearts by 13C-NMR and 1H-NMR spectroscopy.

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C-NMR and 1H-NMR spectroscopy. We demonstrate that 13C-NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments. Wistar rat hearts (five beating and four KCl-arrested hearts) were aerobically perfused with 100% enriched [2-(13)C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 +/- 0.003 min(-1) for beating hearts and 0.0741 +/- 0.004 min(-1) for KCl-arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl-arrested hearts are 1.06 +/- 0.06 micromol x min(-1) x mg(-1) and 0.21 +/- 0.02 micromol x min(-1) x g(-1), respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 +/- 1.7% and 8.8 +/- 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2-oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half-time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.

Acetic Acid↗
PubMed1990

31P NMR studies of spinach leaves and their chloroplasts.

An experimental arrangement is described which enables high quality 31P NMR spectra of compressed spinach leaf pieces to be continuously recorded in which all the resonances observed (cytoplasmic and vacuolar Pi, glycerate-3-P, nucleotides) were sharp and well resolved. 31P NMR spectra obtained from intact chloroplasts showed a distinct peak of stromal Pi. An upfield shift of the stromal Pi resonance was associated with a decrease in the external Pi and vice versa. Nucleotides were largely invisible to NMR in intact chloroplasts, whereas the same nucleotides reappeared in a typical 31P NMR spectrum of an acid extract of intact chloroplasts. Perfusion of compressed spinach leaf pieces with a medium containing Pi triggered a dramatic increase in the vacuolar Pi over 12 h. Addition of choline to the Pi-free perfusate of compressed leaf pieces resulted in a steady accumulation of phosphorylcholine in the cytoplasmic compartment at the expense of cytoplasmic Pi. When a threshold of cytoplasmic Pi concentration was attained, Pi was drawn from the vacuole to sustain choline phosphorylation. In spinach leaves, the vacuole represents a potentially large Pi reservoir, and cycling of Pi through vacuolar influx (energy dependent) and efflux pathways is an efficient system that may provide for control over the cytosolic-free Pi and phosphorylated intermediate concentrations. 31P NMR spectra of neutralized perchloric acid extracts of spinach leaves showed well defined multipeak resonances (quadruplet) of intracellular phytate. The question of cytosolic Pi concentration in green cells is discussed.

Adenine Nucleotides↗
PubMed2003

Spectrophotometric and titrimetric determination of nizatidine in capsules.

Four simple and accurate methods are described for the determination of nizatidine (NIZ) in pharmaceutical preparations. The first method is based on the formation of an ion-pair complex between the drug and either of bromocresol purple or picric acid with subsequent measurement of the developed colors at 411 and 400 nm, respectively. The second method depends on the condensation of mixed anhydrides of citric acid/acetic anhydride, with the tertiary amino group of the drug, where the developed color is measured spectrophotometrically at 545 nm. The oxidation of nizatidine by N-bromosuccinimide was utilized as a basis for the titrimetric method for its assay in capsules. The last method depends on the oxidation of nizatidine by ammonium cerium IV sulfate in the presence of perchloric acid with subsequent measurement of the absorbance at 314 nm; this principle is adopted to develop a kinetic method for the determination of NIZ in capsules. All the reaction conditions have been studied. The detection limits were varied from 0.44 to 0.78 microg ml(-1). The proposed methods were successfully applied to the assay of nizatidine in capsules.

Acetic Anhydrides↗
PubMed1987

A translational inhibitor from muscles of diabetic rats: identification as histone H1.

A heat- and acid-stable protein fraction that inhibited peptide chain initiation in rabbit reticulocyte lysates was extracted from frozen, powdered rat skeletal muscles by stepwise trichloroacetic acid precipitation. Streptozotocin-induced diabetes increased the inhibitory activity; this was prevented by insulin therapy. Size-exclusion high-performance liquid chromatography resolved four inhibitory fractions; only one was consistently increased (approximately 2-fold) in muscle extracts from diabetic rats. Polysome profiles of lysates incubated with this fraction indicated peptide chain initiation inhibition. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified inhibitory fraction migrated with apparent Mr 30 and 32 kDa, which on Western blot immunostained with antisera against histone H1/H1(0). Perchloric acid extraction of muscle homogenates yielded approximately twofold more H1 from diabetic than from control rats; yield from diabetics decreased to control values 5 h after subcutaneous insulin injection. Inclusion of detergent during homogenization increased H1 yield more from muscles of control than from diabetic rats and abolished the difference between them. Because H1 affects several biochemical reactions, its facilitated extraction from insulin-deprived tissues can bias interpretation of studies of insulin action.

Animals↗
PubMed2003

The effects of using lactic acid bacteria inoculants in maize silage on the formation of biogenic amines.

Silages from five ripened varieties of silage maize with dry matter contents ranging between 275 and 410 g/kg were prepared in five laboratory experiments. Whole-plant maize was fermented at 22 degrees C and silages were then stored at the same temperature for 4 months. Spontaneously fermented silages were prepared as control variants and compared with silages inoculated with commercial strains of Lactobacillus plantarum, Lactobzcillus buchneri and a mixed preparation Microsil containing L. plantarum, Lactobacillus casei, Enterococcus faecium and Pediococcus pentosaceus. The starter cultures were applied at doses 5 x 10(5) and 5 x 10(6) CFU/g of chopped maize. Seven biogenic amines and polyamines were extracted from silages with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Common chemical criteria of silage quality were also determined. All three inoculants, mainly at the higher dose, decreased significantly contents of tyramine, putrescine and cadaverine, three undesirable amines occurring at the highest levels. L. plantarum was the most effective. Contents of histamine and tryptamine were low in all experimental silages. Also relatively low were levels of polyamines spermidine and mainly of spermine.

Animals↗
PubMedDate not supplied

Nuclear magnetic resonance detection of increased cortical GABA in vigabatrin-treated rats in vivo.

1H Nuclear magnetic resonance ([1H]NMR) spectroscopy was used to detect elevation of gamma-aminobutyric acid (GABA) in rat brain after administration of the antiepileptic drug vigabatrin (VGB). Rats were treated for 3 weeks with VGB added to their drinking water to deliver a dose of 250 mg/kg body weight per day. NMR spectroscopy was performed noninvasively in vivo, and a GABA concentration of 6.0 +/- 2.3 mmol/kg wet weight (mean +/- SD, n = 5) was measured. GABA could not be detected in control animals in vivo. Postmortem GABA levels of 1.3 +/- 0.5 and 4.5 +/- 1.0 mmol/kg (mean +/- SD, n = 5) were measured in perchloric acid extracts of frozen brain from control and treated animals, respectively. Noninvasive measurement of increased cerebral GABA should allow detailed studies of the pharmacology of GABA-increasing drugs in vivo. With future developments, these measurements may be feasible in human subjects.

Aminocaproates↗
PubMed1982

Purification of carcinoembryonic antigen by affinity chromatography with anti-alpha 1-acid glycoprotein.

Purification of radiolabeled carcinoembryonic antigen (CEA) preparations by affinity chromatography with anti-AG bound to Sepharose was attempted, since an immunological similarity between AG (alpha 1-acid glycoprotein) and a portion of CEA had been noted. When 125I-CEA was purified in this manner, the fraction which did not bind to the column showed decreased reactivity with either anti-AG or anti-CEA. The retained fraction showed enhanced reactivity with both anti-AG and anti-CEA. The yield of purified CEA increased when the CEA preparation was allowed to react with the anti-AG column overnight. Purification of CEA from tumor tissue was performed by affinity chromatography. A perchloric acid (PCA) extract from cancer tissue was mixed with antiserum against CEA to give an immune complex, and a CEA-reactive fraction obtained by PCA extraction. The CEA-reactive fraction was eluted from a Sephadex G-200 column, and final purification was by anti-AG chromatography. When purified CEA was applied to a Sephadex G-200 column with carrier protein after labeling with 125I, the eluted radioactivity was found only in the 180,000 dalton fraction. Almost all the radioactivity was precipitated from the labeled protein by either anti-AG or anti-CEA. Purification of CEA is possible by affinity chromatography with anti-AG bound to Sepharose.

Animals↗
PubMed1977

The perchlorosoluble proteins of the serum of the rainbow trout (Salmo gairdnerii Richardson): albumin like and hemoglobin binding fraction.

1. The main perchlorosoluble fraction of rainbow trout serum has some physico-chemical characters kindred to those of human serum albumin (low molecular weight, solubility with ammonium sulfate, electrophoretic mobility, no glycoproteinic staining). 2. However, on account of obvious differences (heterogeneity and existence of various phenotypes, lack of bromophenol blue or bilirubin binding, low concentration, solubility in perchloric acid), the term "para-albumin" seems more suitable to name this compound. 3. The perchlorosoluble fraction binds hemoglobin causing an increase of peroxidasic activity. But, unlike to human haptoglobin, hemoglobin binding is partial, reversible and labile.

Animals↗
PubMed1978

The presence of guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine in brine shrimp embryos.

Acid-soluble extracts of dormant embryos of the brine shrimp, Artemia salina, contain small amounts of two previously undescribed dinucleotides which we have identified to be guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine. These compounds each comprise about 0.03% of the dry weight of the encysted embryos and are related chemically to guanosine 5'-triphospho-5'-guanosine and guanosine 5'-tetraphospho-5'-guanosine which have been shown previously to be major constituents of the nucleotide pool of Artemia cysts. These new dinucleotides were purified from perchloric acid extracts of dormant cysts by ion exchange column chromatography and identified by means of chemical, spectrophotometric, and enzymatic analyses compared to commercially available compounds. The possible role of these new compounds in nucleotide and nucleic acid metabolism in Artemia embryos is discussed.

Adenosine↗
PubMed1997

The substitution of a single amino acid residue (Ser-116 --> Asp) alters NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis into a glucose dehydrogenase with dual coenzyme specificity.

Glucose-fructose oxidoreductase (GFOR, EC 1.1.1.99.-) from the Gram-negative bacterium Zymomonas mobilis contains the tightly bound cofactor NADP. Based on the revision of the gfo DNA sequence, the derived GFOR sequence was aligned with enzymes catalyzing reactions with similar substrates. A novel consensus motif (AGKHVXCEKP) for a class of dehydrogenases was detected. From secondary structure analysis the serine-116 residue of GFOR was predicted as part of a Rossmann-type dinucleotide binding fold. An engineered mutant protein (S116D) was purified and shown to have lost tight cofactor binding based on (a) altered tryptophan fluorescence; (b) lack of NADP liberation through perchloric acid treatment of the protein; and (c) lack of GFOR enzyme activity. The S116D mutant showed glucose dehydrogenase activity (3.6 +/- 0.1 units/mg of protein) with both NADP and NAD as coenzymes (Km for NADP, 153 +/- 9 microM; for NAD, 375 +/- 32 microM). The single site mutation therefore altered GFOR, which in the wild-type situation contains NADP as nondissociable redox cofactor reacting in a ping-pong type mechanism, to a dehydrogenase with dissociable NAD(P) as cosubstrate and a sequential reaction type. After prolonged preincubation of the S116D mutant protein with excess NADP (but not NAD), GFOR activity could be restored to 70 units/mg, one-third of wild-type activity, whereas glucose dehydrogenase activity decreased sharply. A second site mutant (S116D/K121A/K123Q/I124K) showed no GFOR activity even after preincubation with NADP, but it retained glucose dehydrogenase activity (4.2 +/- 0.2 units/mg of protein).

Amino Acid Sequence↗
PubMed1982

A modified Schayer procedure for the estimation of histidine decarboxylase activity: its application on tissue extracts from gastric mucosa of various mammals.

The question of whether the gastric mucosa of mammals other than rats, mice and hamsters contains an acid (specific) histidine decarboxylase was re-investigated by using a modification of the classical isotope dilution method of Schayer. Its reliability was tested regarding sensitivity, specificity, precision and accuracy, applying criteria recommended by the International Federation of Clinical Chemistry. The assay is now suitable for measuring rather large series of samples. The suitability of several blanks for detecting histidine decarboxylase activity was investigated as a special problem of accuracy. The semicarbazide blank was found to be as reliable as the heating blank, the blank with tissue extract pretreated with perchloric acid or an incubation mixture without radio-labelled histidine. Reaction kinetics of histamine formation were linear over an incubation period of at least 3 h. Histidine decarboxylase activity as a rather low pH and substrate concentration, which is characteristic for the acid (specific) histidine decarboxylase, was demonstrated inthe gastric mucosa of human subjects, dogs, rabbits and guinea-pigs, always using the same incubation conditions for all species investigated. The highest enzymic activity was present in the oxyntic mucosa, but could be measured also in other parts of the stomach.

Animals↗
PubMed1995

Rapid radioassay for metabolites of adenosine and deoxyadenosine in erythrocytes.

A radioassay has been developed to quantify the uptake and initial metabolism of adenosine (Ado) or deoxyadenosine (dAdo) by human erythrocytes. Cell suspension and [3H]Ado are mixed at 3-s intervals with a novel dual-syringe apparatus, and uptake and metabolism of Ado is stopped by centrifuging the cells through a dibutylphthalate layer into perchloric acid. The neutralized cell extract is analyzed by two-dimensional chromatography on poly(ethyleneimine)-cellulose plates by two procedures using combinations of solvents optimised for the separation of nucleosides and nucleobases, and for nucleotides derived from the exogenous [3H]Ado.

Adenosine↗
PubMed1986

[Protein determination in urine--a critical review].

Seven methods for protein determination in urine were systematically checked, viz. turbidimetric assay with benzethonium chloride, turbidimetry with trichloroacetic acid of various concentrations, micellar complex formation with Coomassie Brilliant Blue G-250, formation and gel filtration of protein complexes with copper, reaction of protein tannin compounds with iron, and biuret reaction after different precipitation procedures. The evaluation was mainly based on the following criteria: identical reaction with various proteins, stable and linear absorbance with concentration, complete recovery, and greatest possible freedom from interferences. On this basis, the biuret reaction must now be regarded as the method of choice. Albumin and gamma-globulins are reliably estimated after precipitation by perchloric acid or by an ethanol-diethylether mixture, whereas all proteins, including uromucoid, may be measured after precipitation by Tsuchiya's reagent. Interference from urinary pigments can be avoided by reading versus a sample blank.

Benzethonium↗