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1.
A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [14C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [14C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.Special issue dedicated to Dr. O. H. Lowry. 相似文献
2.
The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of 14CO2 release from positionally labelled [14C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated 14CO2 had no significant effect on the ratio of 14CO2 release from specifically labelled [14C]substrates, or on the metabolism of [U-14C]glucose by the cells. Although the amount of 14CO2 captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of 14CO2 released from different positionally labelled [14C]glucose and [1-14C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [14C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of 14CO2 generated intracellularly during oxidation of [1-14C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [14C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of 14CO2 by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of 14CO2 generated by metabolism. 相似文献
3.
The present study was initiated to determine if algal dietary fibers (DF) bind the carcinogen N-[methyl-14C]-nitrosodimethylamine (DMNA) in vitro and how bioaccumulation of orally-given carcinogen is affected by a diet containing algal DF. Eight kinds of algal DF, including
powdered fronds of Laminaria religiosa (LRP) and agar (from Gracilaria verrucosa) were used in the in vitro test. Cellulose powder (CP) was used as a control DF. In vitro binding rates of DMNA by CP, LRP and agar were 0.28%, 0.65% and 0.21% of the initial dose, respectively. Rats fed a diet
containing 2% LRP or 2% agar were examined at 3 or 24 h after dosing. There was reduced retention of the orally-ingested DMNA
in the liver, possibly because of reduced DMNA-absorption from the intestinal tract earlier than 3 h after dosing. Binding
rates of DMNA by algae were neither related to the DF values nor to the extent of reduction of DMNA-absorption from the intestinal
tract. 相似文献
4.
Eliane Fustec Eric Chauvet Gilbert Gas Daniel Lacaze Michèle Escautier 《Hydrobiologia》1988,159(2):169-176
The fate of lignin in water and sediment of the Garonne river (France) and of a pond in its floodplain was examined using specifically labeled [14C-lignin] lignocelluloses. No significant differences appeared in the mineralization rate of alder, poplar or willow [14C-lignin] in running water samples. Conversion of total radioactivity to 14CO2 ranged between 18.7% and 24.4% after 120 days of incubation. Degree of 14C-labeled lignin mineralization in standing water and sediments was clearly lower, especially in submerged sediments, and was correlated with oxygen supply. After 60 days of incubation 3.3% to 7.9% of the 14C-labeled lignin was recovered in water samples as dissolved organic carbon originating from microbial metabolism. In water extracts from sediment the percentage of dissolved organic 14C was only 0.4% to 1.3% of the applied activity. In the humic fraction extracted from sediments it did not exceed 4.4% which was much lower than in soils. No significant difference appeared between river and pond conditions for humic substances formation. 相似文献
5.
Louise Reyneke Vivienne Ann Russell Joshua Joachim Fransua Taljaard 《Neurochemical research》1992,17(11):1143-1146
This study has shown that neurotensin (NT) increases the electrically stimulated release of [3H]DA to a similar extent in all but the extreme caudolateral area of the rat nucleus accumbens and appears to modulate DA release equally in the medial and lateral zones of this brain area. The simultaneous release of ACh was not significantly affected by NT. 相似文献
6.
To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-13C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of13C derived from [1-13C]glucose into each amino acid using13C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from13C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-13C]GABA were larger than that from [2,3-13C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from13C-amino acids, except13C-alanine, was much smaller than those in the brain and spinal cord. In the serum,13C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-13C]glucose greatly reduced and the intensities of resonances from [3-13C]lactate, [3-13C]alanine, [2, 3, 4-13C]GABA and [2-13C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-13C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of13C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR
nuclear magnetic resonance
- GABA
-aminobutyrate
- GFAP
glial fibrillary acidic protein
Special issue dedicated to Dr. Bernard W. Agranoff. 相似文献
7.
Steven B. Levery 《Glycoconjugate journal》1991,8(6):484-492
Several properties of the exchangeable amide protons of the ganglioside GM2 were studied in detail by1H-NMR spectroscopy in fully deuterated dimethylsulfoxide [2H6]DMSO/2% H2O, and compared with data obtained for the simpler constituent glycosphingolipids GA2 and GM3. In addition to chemical shifts,3
J
2,HN coupling constants, and temperature shift coefficients, the kinetics of NH/2H chemical exchange were examined by following the disappearance of the amide resonances in [2H6]DMSO/2%2H2O. The results included observation of an increase in half-life of theN-acetylgalactosamine acetamido HN by more than an order of magnitude in GM2 compared to GA2, attributable to the presence of the additionalN-acetylneuraminic acid residue. Additional one-dimensional dipolar cross relaxation experiments were also performed on nonexchangeable protons of GM2. The results of all of these experiments support a three-dimensional model for the terminal trisaccharide in which a hydrogen bond is formed between theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group. The interaction is proposed to be of the -acceptor type, a possibility which has not yet been explored in the literature on carbohydrates. The proposed model is discussed in comparison with that of Sabesanet al. (1984,Can J Chem
62: 1034–45), and the models of GM1 proposed more recently by Acquottiet al. (1990,J Am Chem Soc
112:7772–8) and Scarsdaleet al. (1990,Biochemistry
29:9843–55). 相似文献
8.
Dan Gibson Yonit Kasherman Dina Kowarski Inna Freikman 《Journal of biological inorganic chemistry》2006,11(2):179-188
In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl2(13CH3NH2)2] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear
single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release
of 13CH3NH2 but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds
are formed and Pt–NH3 bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C]
HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best
method for characterizing the different species in solution. However, the intensity of the peaks in the 15NH3–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity,
and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary
techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum
complexes with sulfur-containing ligands. 相似文献
9.
Asakawa C Ogawa M Kumata K Fujinaga M Kato K Yamasaki T Yui J Kawamura K Hatori A Fukumura T Zhang MR 《Bioorganic & medicinal chemistry letters》2011,21(8):2220-2223
Sorafenib (Nexavar, BAY43-9006, 1) is a second-generation, orally active multikinase inhibitor that is approved for the treatment of some cancers in patients. In this Letter, we developed [11C]1 as a novel positron emission tomography (PET) probe, and evaluated the influence of ABC transporters-mediated efflux on brain uptake using PET with [11C]1 in P-glycoprotein (P-gp)/breast cancer resistance protein (Bcrp) knockout mice versus wild-type mice. [11C]1 was synthesized by the reaction of hydrochloride of aniline 2 with [11C]phosgene ([11C]COCl2) to give isocyanate [11C]6, followed by reaction with another aniline 3. Small-animal PET study with [11C]1 indicated that the radioactivity level (AUC0-60 min, SUV × min) in the brains of P-gp/Bcrp knockout mice was about three times higher than in wild-type mice. 相似文献
10.
We have established a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of the activator protein which stimulates the enzymic hydrolysis of GM1 (GM1-activator) in human urine. The level of GM1-activator in 19 normal, adult urine samples was estimated to be 370.7±33.2 ng/ml. The amounts of GM1-activator excreted in 24 h were estimated to be between 0.28 and 1.1 mg. The coefficient of variation for this method is 4.3% for the intra-assay and 14.4% for the inter-assay. Urine samples, without purification, can be used directly for the ELISA. 相似文献
11.
Ognen A. C. Petroff Alessandro P. Burlina Joel Black James W. Prichard 《Neurochemical research》1991,16(11):1245-1251
This study explored the utility of1H and13C magnetic resonance spectroscopy to study a standard synaptosomally enriched fraction (P2 pellet) made from rat cerebrum. The preparations contained high concentrations of N-acetylaspartate and -aminobutyric acid and low concentrations of glutamine, indicating that they were in fact rich in neuronal cytosol. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert [1-13C]glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions in 30 minutes. The minimum mean glycolytic rate was 0.8 mM glucose/min and the flow through the tricarboxylic acid cycle was equivalent to 0.2 mM glucose/min.Abbreviations ppm
parts per million (chemical shift scale)
- NMR
nuclear magnetic resonance
- GABA
-aminobutyric acid
- PBS
phosphate-buffered normal saline solution
- TSP
3-trimethylsilylpropionate
During the performance of these studies Dr. A.P. Burlina was on leave from Instituto di Clinica delle Malattie Nervose e Mentali, University of Padua, Padua, Italy. 相似文献
12.
A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C18 cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar GM3-ganglioside is simple and rapid relative to previously described methods. Recovery of GM3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (GM3 synthase; ST-1), the transfer of [14C] sialic acid from CMP-[14C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations GM3
GM3-ganglioside
- II3NeuAc-LacCer
NeuAc2-3Gal1-4Glc1-1Cer
- GD1a
GD1a-ganglioside, IV3NeuAc, II3NeuAc-GgOse4Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer
- GD3
GD3-ganglioside, II3(NeuAc)2LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer
- GgOse4Cer
asialo-GM1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer
- FucGMI
fucosyl-GMI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer
- ST-1
GM3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase
- LacCer
lactosylceramide, Gal1-4Glc1-1Cer
- CMP-NeuAc
cytidine 5-monophospho-N-acetylneuraminic acid
- PC
phosphatidylcholine
- PMSF
phenylmethylsulfonyl fluoride 相似文献
13.
The objectives were to test the null hypotheses that (1) citrate, maltolate, and fluoride do not significantly influence oral Al bioavailability, C(max) or T(max) at an Al dose relevant to drinking water exposure; and (2) Al citrate and maltolate are absorbed intact from the gastrointestinal tract. Male Fisher rats were given 1ml of solution intra-gastrically containing 1 nCi (26)Al (65nmol total Al) as the Al(3+) ion, or as complexes with (14)C-citrate, (14)C-maltolate or fluoride, during concurrent (27)Al iv infusion. Blood was repeatedly collected for serum (26)Al, total Al and (14)C quantification. Absorption parameters were estimated using WinNonlin. Al bioavailability, C(max) and T(max) from the ion, citrate, maltolate, and fluoride were 0.29+/-0.11%, 0.61+/-0.31%, 0.50+/-0.25%, and 0.35+/-0.10%; 659+/-195, 1073+/-250, 881+/-356, and 880+/-295fg/ml; and 1.2+/-0.9, 1.0+/-1.1, 1.3+/-1.0, and 1.0+/-0.9h (X+/-SD) respectively. Serum (14)C was approximately 100 times higher than (26)Al. The results suggest a non-significant enhancement of oral Al bioavailability by citrate and maltolate, some Al complex dissociation in the GI tract, and less absorption of Al than citrate or maltolate. The presence of citrate, maltolate and fluoride, at a similar molar concentration to Al, would not be expected to greatly influence Al absorption from drinking water. 相似文献
14.
Yasuhiro Hashimoto Mikiko Abe Akemi Suzuki Kentaro Iwasaki Tamio Yamakawa 《Glycoconjugate journal》1985,2(3-4):255-265
Genetic polymorphism in the expression of the GM1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of GM1(NeuGc) synthetase, UDP-galactose:GM2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F1 mice, and the female F1 mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.Abbreviations NeuGc
N-glycolylneuraminic acid
The ganglioside nomenclature is based on the system of Svennerholm, J Neurochem (1963) 10:613-23. The sialic acid species present is shown in parentheses after the ganglioside abbreviation. 相似文献
15.
L-[1-13C] phenylalanine breath tests (PheBTs) have been used to determine the hepatocyte functional capacity of patients. This study investigated the relationship between the PheBT parameter 13C excretion rate constant (PheBT-k) and activity of the phenylalanine metabolic rate-limiting enzyme phenylalanine hydroxylase (PAH) in rat liver. We noted that the time-course curves of 13C excretion presented as a single peak, which appeared 2 min after administration of L-[1-13C] phenylalanine (13C-Phe). 13C excretion during exhalation can be divided into a slow phase and a rapid phase. The PheBT-k in rats with carbon tetrachloride acute liver injury was.significantly lower than that of control rats. The rapid phase 13C disposition constants of the acute liver injured rats did not differ from that of the controls. The peak value of 13C abundance in the breath of the acute liver injured rats was markedly higher than that of the control group. Total liver PAH activity in the acute liver injured rats was significantly lower than that in the control group. PheBT-k was highly correlated with the total activity of liver PAH (r = 0.92, P < 0.001). The present findings indicate that PheBT results reflect PAH activity levels. The PheBT-k parameter is a sensitive index that can be used to evaluate PAH function in the liver. In addition we demonstrated that the rodent model used in this study is a valuable tool for basic research studies of the breath test. 相似文献
16.
Jürgen Roth 《Glycoconjugate journal》1985,2(3-4):315-321
Cholera toxin bound to particles of colloidal gold was used to investigate by electron microscopy the binding of the toxin in human duodenum. Cholera toxin binding was detected only in the apical (brush border) plasma membrane domain suggesting that the ganglioside GM1 is absent from the basolateral plasma membrane domain. Intracellularly, toxin binding became detectable in thetrans side of the Golgi apparatus. Labeling of endosomes may indicate that the non toxin-occupied GM1-ganglioside becomes internalized. 相似文献
17.
The effects of adding D2SO4, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D2O were investigated at 23 °C by plotting 13C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. 13C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D2SO4 medium. The C-1 of the α anomer showed a slightly higher response to increasing D+ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule. 相似文献
18.
Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl-
-aspartate receptors following multiple intracerebroventricular injections of [
-Pen2,
-Pen5]enkephalin and [
-Ala2, Glu4]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [
-Pen2,
-Pen5]enkephalin and [
-Ala2, Glu4]deltorphin II, the selective agonists of the δ1- and δ2-opioid receptors, on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-
-aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [
-Pen2,
-Pen5]enkephalin or [
-Ala2, Glu4]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [3H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [3H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [
-Pen2,
-Pen5]enkephalin treated mice. In [
-Ala2, Glu4]deltorphin II treated mice, the binding of [3H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the Bmax value of [3H]MK-801. It is concluded that tolerance to δ1- and δ2-opioid receptor agonists is associated with up-regulation of brain N-methyl-
-aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl-
-aspartate receptors antagonists block tolerance to the analgesic action of δ1- and δ2-opioid receptor agonists. 相似文献
19.
Malcolm J. Thompson Mark F. Feldlaufer Ruben Lozano Huw H. Rees William R. Lusby James A. Svoboda Kenneth R. Wilzer 《Archives of insect biochemistry and physiology》1987,4(1):1-15
Following injection into female Manduca sexta pupae, [14C]cholesterol is converted to a radiolabeled C21 nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C21-steroid conjugates). 相似文献
20.
Full geometric optimization of endo-tricyclo[3.2.1.02,4]oct-6-ene (endo-TCO) by ab initio and DFT methods allowed us to investigate the structure of the molecule. The double bond is endo-pyramidalized and its two faces are no longer found to be equivalent. The exo face of the double bond has regions with far more electron density (qi,HOMO) and more negative electrostatic potential. The endo-TCO-Br2 system was investigated at the B3LYP/6-311+G** level and the endo-TCO···Br2(exo) molecular complex was found to be relatively more stable than the endo-TCO···Br2(endo) complex. The cationic intermediates of the reaction were studied by ab initio and DFT methods. The bridged exo-bromonium cation(I) is relatively more stable than the endo-bromonium cation(II). An absolute exo-facial selectivity should be observed in the addition reaction of Br2 to endo-TCO, which is caused by steric and electronic factors. The nonclassical rearranged cation IV was found to be the most stable ion among the cationic intermediates and the ionic addition occurs via the formation of this
cation. The mechanism of the addition reaction is also discussed. 相似文献