Uniformly labeled

Synthetic chemical approaches to the preparation of carbon-14 labeled materials iavolve a number of basic building blocks prepared from barium [ CJ-carbonate (2). These are carbon [ C]-dioxide [ CJ-acetjlene [U— C]-ben2ene, where U = uniformly labeled [1- and 2- C]-sodium acetate, [ C]-methyl iodide, [ C]-methanol, sodium [ C]-cyanide, and [ CJ-urea. Many compHcated radiotracers are synthesized from these materials. Some examples are [l- C]-8,ll,14-eicosatrienoic acid [3435-80-1] inoxn. [ CJ-carbon dioxide, [ting-U— C]-phenyhsothiocyanate [77590-93-3] ftom [ " CJ-acetjlene, [7- " C]-norepinephrine [18155-53-8] from [l- " C]-acetic acid, [4- " C]-cholesterol [1976-77-8] from [ " CJ-methyl iodide, [l- " C]-glucose [4005-41-8] from sodium [ " C]-cyanide, and [2- " C]-uracil [626-07-3] [27017-27-2] from [ " C]-urea. All syntheses of the basic radioactive building blocks have been described (4). 

Microbiological procedures which exploit the ability of bacteria and photosynthetic algae to incorporate exogenous labeled precursors such as 14CO2, SO%, and 32pQ3- [ can be used to label complex molecules in cells such as proteins (qv) and nucleic acids (qv), which are then processed to give labeled constituents such as uniformly labeled C-amino acids, C-nucleotides, C-fipids, LS-amino acids, etc (8). 

TABLE 1. Chemical oxidation of methionine in HL-60 cell proteins previously uniformly labeled with [35S]methionine ... 

Rules for designating isotopic substitution and labelling are given in [13] (Section H). Parentheses indicate substitution square brackets indicate labelling. The locant U indicates uniform labelling. 

Figure 1. Immunofluorescent labeling of dystrophin in the Xp21 muscular dystrophies. In normal muscle, clear uniform labeling is present at the membrane of each muscle fiber. In Becker muscular dystrophy (BMD), there is inter- and intrafiber variation in labeling intensity. In Duchenne muscular dystrophy (DMD), most fibers are devoid of labeling (note, however, that in most biopsies occasional fibers exhibit weak labeling). In the biopsy from a manifesting carrier, some fibers show normal labeling and others are negative. In the former, the normal X-chromosome is active while in the latter the abnormal X-chromosome is active.

The Preparation of Uniformly Labeled C-2,7-Dichlorodibenzo- -dioxin and C-2,3,7,8-Tetrachlorodibenzo- -dioxin... 

Dichlorodibenzo-p-dioxin was prepared from isotopic potassium 2,4-dichlorophenate uniformly labeled with Ullman conditions gave a 20.5% yield. Small amounts of dichlorophenoxy chlorophenol were removed from the product by extraction with sodium hydroxide before purification by fractional sublimation and recrystallization from anisole. Chlorination of 2,7-dichlorodibenzo-p-dioxin in chloroform solution containing trace amounts of FeCls and 12 yielded a mixture of tri-, tetra-, and pentachloro substitution products. Purification by digestion in boiling chloroform, fractional sublimation, and recrystallization from anisole was effective in refining this product to 92% 2,3,7,8-tetrachloro isomer, which also contained 7% of the tri- and 1% of the penta-substituted dibenzo-p-dioxin. Mass spectroscopy was used exclusively to monitor the quality of the products during the synthesis. 

Uniformly labeled 2,4-dichlorophenol- C (purchased from New England Nuclear Corp, Boston, Mass.) was used in the tracer preparation. This provided a label at all carbon positions in the dibenzo-dioxin structure. 2,7-Dichlorodibenzo-p-dioxin- C after initial cleanup by fractional sublimation, contained approximately 5% of an impurity, detected by thin layer chromatography (TLC) which gave mass peaks at 288, 290, 292, and 294 in the mass spectrometer, consistent with a trichloro-hydroxydiphenyl oxide. This is probably the initial condensation product of the Ullman reaction and is most likely 2-(2,4-dichlorophenoxy)-4-chlorophenol. It was removed easily by extractions with aqueous... 

Uniformly labeled C-8-D with a specific activity of 2.99 juc/mg was administered orally to pregnant females at 2 /xg/kg/day from 6-15 days of gestation. Three females were sacrificed on alternate days during days 6-20 of pregnancy. Triplicate samples of fetus, placenta, blood, brain, abdominal fat, and sartorius muscle were procured from each female. The samples were dissolved in 1 ml of Soluene (Packard Instruments) to which 15 ml of Aquasol were added. Each sample vial was counted for 30 min in a Nuclear Chicago Mark I liquid scintillation counter. 

TAetection of the highly potent impurity, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), necessitated an environmental assessment of the impact of this contaminate. Information was rapidly needed on movement, persistence, and plant uptake to determine whether low concentrations reaching plants, soils, and water posed any threat to man and his environment. Because of the extreme toxicity of TCDD, utmost precautions were taken to reduce or minimize the risk of exposure to laboratory personnel. Synthesis of uniformly labeled C-TCDD by Muelder and Shadoff (I) greatly facilitated TCDD detection in soil and plant experiments. For unlabeled experiments it seemed wise to use only small quantities of diluted solutions in situations where decontamination was feasible and to rely on the sensitivity afforded by electron capture gas chromatography... 

Single dermal doses of 50 mg/kg tri-ort/ o-cresyl-[uniformly labeled 14C-phenyl]phosphate (TOCP) were applied to preclipped, unprotected, 10-cm2 areas of skin in male cats (Nomeir and Abou-Donia 1986). 

A study with a dog exposed to an occluded dermal dose of TOCP labeled with radioactive phosphorus provides limited evidence that organophosphate esters in hydraulic fluids may be widely distributed after dermal absorption (Hodge and Sterner 1943). Similar widespread distribution of radioactivity among tissues was observed in male cats after dermal exposure to [uniformly labeled 14C-phenyl]TOCP (Nomeir and Abou-Donia 1986). Tissues and fluids with the highest concentrations of radioactivity in these studies included the bile, gall bladder, urinary bladder, liver, kidney, and fat, thus suggesting that TOCP and metabolites are somewhat preferentially distributed to these tissues. 

In male cats, peak concentrations of radioactivity in most tissues and fluids were observed 24 hours after application of 50 mg/kg [uniformly labeled 14C-phenyl]TOCP to unprotected areas of skin on the back of the neck (Nomeir and Abou-Donia 1986). Peak concentrations of radioactivity (reported in parentheses in... 

A recent series of experiments with cats, chickens, or rats exposed to [uniformly labeled 14C-phenyl]-TOCP shows that a complex array of oxidized and dearylated metabolites are found in excreta and various tissues including the liver, kidney, testis, and brain (Abou-Donia et al. 1990a, 1990b Nomeir and Abou-Donia 1986 Somkuti and Abou-Donia 1990). Cats and chickens, like humans, are sensitive to TOCP-induced delayed neuropathy (Baron 1981). A similar array of oxidized and dearylated derivatives of tri-para-cresyl phosphate (but no cyclic metabolites) were identified by mass spectrometry in the urine and... 

With advances in multidimensional NMR technology and the development of methods for uniform labeling of proteins with 15N and 13C, direct NMR studies on unfolded states of full-length proteins, rather than protein fragments, became possible. These studies were aided by the advent of high-field NMR spectrometers, which provide the necessary dispersion and sensitivity. High sensitivity is a critical factor in the study of unfolded and partly folded proteins, since NMR experiments must often be performed at very low concentrations to prevent aggregation. 

Recently, numerous studies reported the application of homonuclear and heteronuclear selective recoupling schemes on uniformly labelled ligand interacting with membrane receptors. The polarization exchange curves were fitted with the two-spin model and showed that it is possible to determine intemuclear distances up to 4.5 A.118... 

Frequency-selective REDOR (fsREDOR) is a very powerful technique developed for the study of 13C and 15N uniformly labeled peptides or proteins [92]. The basic idea of this technique is to combine REDOR and soft n pulses to recouple a selected 13C-15N dipole-dipole interaction in a multiple-spin system. Usually one could use Gaussian shaped pulses to achieve the required selective n inversions. Other band selective shaped pulses have been developed for a more uniform excitation profile [93]. In its original implementation, fsREDOR was used to extract the intemuclear distances of several model crystalline compounds [92], In the past few years, this technique has proven to be very useful for the study of amyloid fibrils as well. For the Ure2p10 39 fibril samples containing 13C and 15N uniformly... 

Fig. 1.9 [ 5 N. HJ-HSQC spectrum of 15N-uniformly labeled SH3 domain (A) and of a sample selectively...

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