Radioactive precursor

In the last column of Table 7.1, the most popular radioactive precursor nuclide is given together with the nuclear decay process (EC = electron capture, = beta decay) feeding the Mossbauer excited nuclear level. 

After 30 to 60 min incubation at 37°, 20- to 40-/d aliquots from each reaction mixture are spotted on 3MM paper discs that are dropped into 10% ice-cold TCA and processed according to the hot TCA procedure (see later). If the translational product is to be quantified only immunologically, the radioactive precursor ( [14C] phenylalanine) is replaced by nonradioactive phenylalanine. Examples of experiments in which the preceding protocol has been used are shown in Fig. 12.3A.B. 

This procedure to test the levels of translation using a radioactive precursor was originally described by Mans and Novelli (1960). At the end of the incubation, aliquots of the reaction mixtures are spotted onto 3MM paper discs, which are immediately dropped into 10% ice-cold TCA and processed as described for the cold TCA procedure, except that the three washes at room temperature in 5% TCA are preceded by a 10-min wash at 90°. 

It is difficult to reconcile the unique chemical structure of tetrodotoxin with that of an animal product. Its structure is not related to that of other animal products by any readily recognized biosynthetic scheme. It is not a terpenoid, not obviously formed from amino acid or carbohydrate units, and apparently not constructed from acetate or propionate units. Nor does it resemble any of the various plant alkaloid patterns. It thus appears to be a very unlikely animal product to result from known biogenetic pathways. In this connection the metabolic incorporation of radioactive precursors using torosa and ]C. granulosa salamanders was studied by Shimizu et al. (47). They observed significant isotopic incorporation into amino acids and steroid metabolites, but they found no such incorporation associated with tetrodotoxin. 

There is some evidence for the occurrence of a glycolyl group at 0-4 of Neu5Ac in serum and submandibular-gland glycoproteins from the horse, based on biosynthetic studies with radioactive precursors,33 and chemical and t.l.c. analysis of the sialic acid ester groups.74... 

Although preparation of H-L-Phe(3,5-3H2,4-NH2)-OH (31) and its incorporation into Z-Ala-Ala-Phe(3,5-3H2,4-NH2)-OH has been reported, its general applicability is very limited J30 Carrying out on a radioactive precursor the extensive protection schemes necessary to minimize side reactions, and the multiple elongation steps required in a standard peptide... 

Cell kinetics is defined as the measurement of time parameters m biological systems. Traditionally, this has involved the use of radioactive precursors of DNA, such as tritiated thymidine (3HTdR), and autoradiography to detect their incorporation into DNA. This technique has provided detailed knowledge of cell kinetics in both in vitro and in vivo experimental systems. The technique, however, is time consuming and arduous and is not readily applicable to human tumor research because of ethical problems involved in incorporation of a radioisotope into DNA. 

I he atomic wcighi varies because of natural variations in the isotopic composition of the element, caused by the various isotopes having different origins - I h is the end product of the thorium decay scries, while Ph and " Pb arise Irom uranium as end products of the actinium and radium series respectively. Lead-204 has no existing natural radioactive precursors. Electronic configuration l.v 2s lfc22/j"3v 3//,3i/l"4v- 4/, 4l/" 4/ IJ5v- 5/ "5t/l"bv />-. Ionic radius Pb I.IX A. Pb 1 0.7(1 A. Metallic radius 1.7502 A. Covalent radius (ip i 1.44 A. First ionization potential 7.415 cV second. 14.17 eV. Oxidation... 

Incorporation of Radioactive Precursors into Lipids. The cultures of surface-adhering cells were exposed for 16 h to either 400 pCi of Hp/C- syo (final specific activity, 50 yCi/ ymol) or 10 pCi of / %/, galactose ( the rate of incorporation was linear during this period). After 16 hr. the radioactive medium was removed and the cultures were washed four times with 0.9 NaCl. The cells were removed from the surface with a rubber policeman and suspended in physiological saline. Lipids were extracted by Bligh and Dyer procedure (26) and analyzed for various lipids according to Neskovic, et al. (27)... 

Longer term assessments of cell toxicity are highly dependent on the relevant toxic end point. They may include measurement of growth competence, apoptosis, and/or necrosis, incorporation of radioactive precursors into essential cellular constituents such as RNA, DNA, and protein and specialized cellular functions. Some examples of the use of cultured cell lines in the study of toxicity effects are shown in Table 2.1. 

The biosynthetic pathways of reserpine and other alkaloids of Rauwalfia have been extensively studied by several authors. Leete (l8, 19) fed DL-[2- C]-tryptophan into Rauwolfia serpentina (3 years old plants) which led to the formation of radioactive ajmaline, serpentine and reserpine (18). Later, he found that radioactive serpentine was labeled solely at C-5 indicating that tryptophan was a direct precursor of the -carboline moiety of this alkaloid (19). Other radioactive precursors have been administered to Rauwolfia plants such as [l- C] acetate (20), [2- C] acetate (21), [2-11 C] alanine (20) and [2-l2 C] glycine (21, 22). All incorporated into ajmaline and reserpine. 

Studies involving timed incubations with drugs, radioactive precursors, hormones, etc. In these instances it is important to harvest the cells promptly without exposure at 37°C to an altered environment for an indefinite period. 

Cells growing directly in dishes (5 cm Petri dishes) may be labelled with a radioactive precursor and fixed in a similar way to cells on coverslips. One ml of diluted liquid emulsion is then added and the material exposed (without the lid) and developed as indicated above. 

Autoradiographic analysis is the only method which can determine the proportion of cells incorporating a radioactive precursor and the site of that incorporation. Thus, tritiated thymidine is incorporated into DNA in the nuclei of those cells in S-phase and tritiated hypoxanthine appears first in the nucleus and later in the cytoplasm of cells with HPRT but not in mutants lacking the enzyme (see 13.2). 

Autoradiography is a technique for locating radioactive compounds within cells it can be conducted with light or electron microscopy. Living cells are first exposed to the radioactive precursor of some intracellular component. The labeled precursor is a compound with one or more hydrogen ( H) atoms replaced by the radioisotope tritium (3H) e.g., [3H]thymidine is a labeled precursor of DNA, and [3H]uridine is a labeled precursor of RNA (Chap. 7). Various tritiated amino acids are also available. The labeled precursors enter the cells and are incorporated into the appropriate macromolecules. The cells are then fixed, and the samples are embedded in a resin or wax and then sectioned into thin slices. 

In an attempt to determine the localization of glycogen in the liver, could there be any problems of interpretation of the electron microscopic autoradiographic images if [3H]glucose were used as the radioactive precursor molecule of glycogen ... 

The incorporation of tritiated thymidine into DNA was frequently used as an assay to identify growth factors and study their effects on cell proliferation. The method involves addition of a radioactive precursor that is incorporated into newly synthesized DNA during the S phase of the cell cycle. Cells are cultured to allow incorporation of the tracer, followed by acid treatment of samples to precipitate DNA contained in cells, and subsequent separation of unincorporated tracer. Special equipment was designed to process increasing numbers of radioactive samples. 

The biosynthesis of GAs was extensively surveyed during the 1960-70s by feeding experiments using Gibberella fujikuroi and radioactive precursors.199 200 The studies on the biosynthesis of GAs in plants were led by Graebe and coworkers.201-203 Recent research progress in the biosynthesis of GAs is described in Section 4.02.3.4. 

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