Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Synthesis of [5'-11C]ribonucleoside and -2'-deoyribonucleoside derivatives from D-ribose

An approach to the synthesis of 2'-deoxy[5'-13C]ribonucleosides was achieved by the coupling reaction of a nucleic acid base derivative with D-[5-13C]ribose derivative (8). Compound 8 was derived from D-ribose (1) by way of methyl 2,3-di-O-benzyl(Bn)-D-ribofuranoside (2), 2,3-di-O-Bn-D-ribose diethyl dithioacetal (3), 2,3-di-O-Bn-D-ribose dibenzyl acetal (4), and 4-aldehydo-2,3-di-O-Bn-D-erythrose dibenzyl acetal (5), which was then successively subjected to Wittig reaction using Ph3P13CH3I-BuLi, highly stereoselective hydroxylation with OsO4 to give 2,3-di-O-Bn-D-ribose dibenzyl acetal (7), debenzylation with H2-Pd/C. The resulting 8 was subjected to coupling reaction with a nucleic acid base to give [5'-13C]ribonucleosides. The products were derived into the corresponding 2'-deoxy[5'-13C]ribonucleoside derivatives by the established manner.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Binding of [3H][D-Ala2, MePhe4, Gly-ol5] enkephalin, [3H][D-Pen2, D-Pen5]enkephalin, and [3H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats

The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [3H]labeled ligands of mu-([D-Ala2, MePhe4, Gly-ol5]enkephalin; DAMGO), delta ([D-Pen2, D-Pen5]enkephalin; DPDPE), and kappa-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [3H]DAMGO, [3H]DPDPE and [3H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was delta- > or = kappa- > or = mu-, with lung parenchyma exhibiting the greatest binding capacity for delta- and mu- receptors compared to the other regions examined. The Kd values showed small differences between ligands and regions tested. The mu- and delta-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, kappa-opioid receptor density was augmented in sensitized lung parenchyma but an increase in Kd values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.     

Effect of amygdaloid kindling on [3H]dopamine and [14C]acetylcholine release from rat prefrontal cortex and striatal slices

The involvement of the dopaminergic (DA) systems in the control of limbic kindled seizures is ill defined. The effects of kindling on DA activity may have been overlooked in the past, because of its subtle unilateral occurrence and/or the variance of the endogenous imbalance of DA activity in normal animals. In the present study rats were screened for their endogenous DA imbalance using amphetamine-induced rotational behaviour. Electrical or sham kindling was applied in the hemisphere with the higher endogenous DA activity. Sections of the bilateral prefrontal cortex and dorsal and ventral striatum were dissected either 2 hours or 21 days after the final seizure and the electrically stimulated release of [3H]DA and [14C]acetylcholine (ACh) determined. Release was also measured in the presence of quinpirole or sulpiride to assess the activity of pre- and postsynaptic DA D2-receptors. Long-term effects of kindling consisted of facilitation of ACh release in the ventral striatum contralateral to the kindled amygdala and bilateral depression of DA release in the prefrontal cortex. Kindling therefore produced area specific changes in neurotransmitter systems giving rise to increased pro-convulsive cholinergic activity in the ventral striatum and decreased anti-convulsive dopaminergic activity in the prefrontal cortex.     

Laminar distribution of nicotinic receptor subtypes in human cerebral cortex as determined by [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in vitro autoradiography

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

The metabolism of [6-3H]1alpha, hydroxycholecalciferol to [6-3H]1alpha,25-dihydroxycholecalciferol in a patient with renal insufficiency

To evaluate whether 1alpha-hydroxycholecalciferol is metabolized to 1alpha,25-dihydroxycholecalciferol in man, [6-3H]1alpha-hydroxycholecalciferol was given intravenously to a patient with renal failure who was maintained daily on 100,000 IU vitamin D and calcium supplements. Using Sephadex LH-20 and high-pressure liquid chromatography, it was clearly demonstrated that 1alpha-hydroxycholecalciferol rapidly disappears from the blood and is metabolized to 1alpha,25-dihydroxycholecalciferol.     

Digestion and absorption rates of [3H]-oleic acid and [14C]-triolein do not differ in rats fed heated (-) and (+) gossypol cottonseed and soybean flours

This study was conducted to compare in vivo the acute effects of heated (+) and (-) gossypol cottonseed flours with those of soybean flour on lipid digestion and absorption in growing rats. Rats were fed by gastric intubation mixed [3H]-oleic acid and [14C]-triolein with heated flours or without flour (control). Lipid digestion and absorption were determined for 6 h after meal intubation. Both radioactivities recovered in gastrointestinal tract were significantly higher in rats fed (+) gossypol cottonseed flour than in all other groups. The majority of both recovered radioactivities was found in stomach contents, then in stomach wall and finally in intestinal wall. The distribution of both radioactivities at different gastrointestinal levels was similar. In stomach contents and wall, [14C]-radioactivity was primarily in triacylglycerols, but was also recovered in free fatty acids and diacylglycerols. In intestinal wall (mucosa + tunica) [3H]-radioactivity was found at greatest levels in free fatty acids, then in triacylglycerols and diacylglycerols. Greatest [14C]-radioactivity was found in triacylglycerols, then in free fatty acids, in diacylglycerols and last in phospholipids in rats fed the three flours. Therefore no quantitative differences in lipid digestion and absorption were observed among the rats fed the three flours. In conclusion, both cottonseed flours slowed lipid digestion and absorption compared with soybean flour and this delay was greater in the rats fed (+) gossypol cottonseed flour than in those fed (-) gossypol cottonseed flour. However, this inhibiting effect was probably too low to induce physiologically important effects on lipid digestion or absorption.     

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.