Duodenal vs. gastric administration of labeled leucine for the study of splanchnic metabolism in humans.
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Low-rate (6 ml/h) intragastric infusion of stable, isotope-labeled amino acids is commonly used to assess the splanchnic handling of amino acids in humans. However, when used in the postabsorptive state, this method yields unreliable plasma isotopic enrichments, with a coefficient of variation >10%. In this metabolic condition, we confirmed in six subjects that an intragastric infusion of L-[(2)H(3)]leucine at 6 ml/h yields an unreliable isotopic steady state in plasma amino acids with a coefficient of variation of 43 +/- 12% (mean +/- SD). In five additional subjects, we assessed the effects of 1) increasing the rate of delivery of a leucine tracer in an isotonic plasmalike solution at 240 ml/h into the gastric site, and 2) changing the site of infusion from gastric to duodenal with this same high rate of delivery. In contrast to the gastric route, and regardless of the rate of delivery, only the intraduodenal route allowed 1) isotopic plasma steady state (i.e., coefficients of variation were <10%: 5 +/- 3%), and 2) reproducible leucine extraction coefficients (22 +/- 5%). We conclude that an infusion site that bypasses the gastric emptying process, i.e., the duodenal route, along with delivery of a plasmalike solution, is necessary to reach isotopic steady state in plasma when labeled leucine is infused into the gastrointestinal tract in the postabsorptive state. (+info)
A simple and efficient method for radiolabeling of preformed liposomes.
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A simple and efficient method for radiolabeling preformed liposomes was developed using hepatobiliary imaging agent (99m)Tc-diisopropyl iminodiacetic acid ((99m)Tc-DISIDA). Chloroform extraction of (99m)Tc-DISIDA from aqueous solutions results in 80% radioactivity in the organic phase due to its lipophilic properties. However, with the presence of reduced glutathione (gamma-Glu-Cys-Gly), chloroform extraction results in only 30% of label in the organic phase because the (99m)Tc-DISIDA complex undergoes reduction decomposition to more hydrophilic species by reaction with glutathione. The incorporation efficiency of the (99m)Tc-DISIDA into the liposomes containing reduced glutathione was greater than 90%. The labeled liposomes were stable up to 24 h in saline and 90% FBS after preparation. Biodistribution studies in mice showed that (99m)Tc labeled liposomes accumulated in liver and spleen at 24 h postinjection, unlike (99m)Tc-DISIDA. Compared to hexamethylpropyleneamine oxime (HMPAO), the (99m)Tc-DISIDA compound is much cheaper and has a longer shelf life when used for liposome labeling. The labeling technique described here could be used for monitoring pharmacokinetic and pharmacodynamic changes of liposomes, and tumor or infection imaging when coupled with targeting antibodies. (+info)
A link between streptomycin and rifampicin mutation.
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Introduction of str A mutations frequently make "male" strains of Escherichia coli permissive to bacteriophage T7; certain rif mutations reverse the permissive effect of strA mutation. Permissiveness of the strA mutation is accompanied by enhanced transcription of bacteriophage T7 genome. Introduction of the nonpermissive rif allele to the permissive strA strain reduces or abolishes the transcription of T7 genome. Thus, a link is implied in the functioning of the ribosome and the RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6). (+info)
Novel mutants of Escherichia coli that accumulate very small DNA replicative intermediates.
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A new group of mutants has been isolated which, during short pulses, incorporate (3-H)thymidine into DNA fragments that are substantially smaller than Okazaki fragments. These small fragments can be chased into DNA of high-molecular-weight, and thus may be precursors in DNA replication, During longer pulses, label also appears in DNA of higher molecular weight, although at an abnormally slow rate. The mutations map at a previously undescribed locus (dnaS) at 72 min on the E. coli chromosome. (+info)
Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation.
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A temperature-sensitive mutant having a lethal mutation in the gene for the beta subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) exhibits an apparent 2- to 3-fold decrease in the rates of both beta and beta' subunit synthesis at the non-permissive temperature, relative to total protein. In contrast, a temperature-sensitive mutant with a lethal mutation in the gene encoding beta' has a 5- to 6-fold increase in the rates of beta and beta' synthesis at 42 degrees. These beta and beta' mutants also exhibit rapid degradation of both subunits at the high temperature. (+info)
Biosynthesis of abscisic acid by the non-mevalonate pathway in plants, and by the mevalonate pathway in fungi.
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The biosynthetic pathways to abscisic acid (ABA) were investigated by feeding [1-(13)C]-D-glucose to cuttings from young tulip tree shoots and to two ABA-producing phytopathogenic fungi. 13C-NMR spectra of the ABA samples isolated showed that the carbons at 1, 5, 6, 4', 7' and 9' of ABA from the tulip tree were labeled with 13C, while the carbons at 2, 4, 6, 1', 3', 5', 7', 8' and 9' of ABA from the fungi were labeled with 13C. The former corresponds to C-1 and -5 of isopentenyl pyrophosphate, and the latter to C-2, -4 and -5 of isopentenyl pyrophosphate. This finding reveals that ABA was biosynthesized by the non-mevalonate pathway in the plant, and by the mevalonate pathway in the fungi. 13C-Labeled beta-carotene from the tulip tree showed that the positions of the labeled carbons were the same as those of ABA, being consistent with the biosynthesis of ABA via carotenoids. Lipiferolide of the tulip tree was also biosynthesized by the non-mevalonate pathway. (+info)
A new method for one-step, no-carrier-added synthesis of cholesteryl 4-[18F]-fluorobenzoate ([18F]-CFB), a radiotracer used in detection of adrenal malignancies.
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PURPOSE: Cholesteryl 4-[(18)F]-fluorobenzoate, a potential radiotracer used for adrenal and ovarian imaging, was prepared in no-carrier-added form from cholesteryl 4-N,N,N-trimethylanilinium trifluoromethanesulfonate. METHODS: The reaction was performed in one step using Kryptofix2.2.2/[(18)F], carbonate as the counter ion and dimethyl sulfoxide as the solvent at 110 degrees C. Purification was performed using commercially available C(18) and Si Sep-Paks. RESULTS: Column purification afforded the desired compound in 75-85 % radiochemical yield (EOS) with a specific activity about 74 KBq/mmole in about 20 minutes, with greater than 95% radiochemical and chemical purity (HPLC and TLC analysis). CONCLUSIONS: This compound was prepared through a novel method which can be easily performed at distant locations from the main radionuclide production centers using Sep-Paks. The biodistribution of this compound in mice was confirmed to be similar to that reported in the literature. (+info)
Synthesis and turnover of basal level guanosine tetraphosphate in Escherichia coli.
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Cultures of escherichia coli growing exponentially in Trisacetate medium were subjected to nutritional shift-up and the pool size of guanosine 5'-3'-diphosphate-3'diphosphate (ppGpp) as well as the rates of protein synthesis and net RNA synthesis were determined. In the shift to a rich medium (glucose plus 19 amino acids plus hypoxanthine) the basal level of ppGpp falls immediately with a decay constant suggesting total inhibition of synthesis; ther is no ppGpp detectable above background for 30 to 40 min. The net rate of RNA synthesis starts to increase within 1 min of the shift-up and has reached its definite postshift value well before the pool of ppGGpp rises above background lvel. In a shift-up from Tris-acetate medium to Tris-glucose medium there is a much slower readjustment of the ppGpp pool size without the transient disappearance of the nucleotide. However, in a shift-up to Tris-acetate plus 5 amino acids, a medium which supports the same growth rate as Tris-glucose medium, a dramatic, transient drop in the ppGpp pool level was observed. Relaxed cells exhibit very similar behavior to strigent cells in the same shift-up. Our data argue strongly against an exclusive role for pGpp in regulating RNA synthesis during niutritional shift-up. The kinetic data of [3H]guanosine uptake into GTP and ppGpp pools were analyzed to determine the rate of pGpp synthesis. This rate was found to be similar during expotential growth in either Tris-acetate medium. During a shift-down from Tris-glucose to Tris-acetate medium the rate of ppGpp syntesis fell by a factor of 1.5 to 2 and the turnover rate is reduced 6- to 8-fold, suggesting that the expansion in the ppGp pool during shift-down canot be due to derepression of synthesis. (+info)