General method to label antisense oligonucleotides with radioactive halogens for pharmacological and imaging studies

Evaluation of oligonucleotides for biomedical applications requires different in vivo and in vitro approaches (pharmacokinetics, biodistribution, macro- and microimaging, metabolism,.), that are performed with different radioisotopes according to the temporal and spatial resolution needed. A method to introduce radioactive isotopes of halogens (fluorine, bromine, and iodine) in a small and stable molecule has been developed. Radiosynthons can then be conjugated with any given oligonucleotide in one step to create the appropriate radiotracer. This general radiolabeling procedure for oligonucleotides is efficient to synthesize (18)F-, (76)Br-, and (125)I-oligonucleotides for biological needs. Applications of the method to biodistribution, metabolism, in vivo and ex vivo imaging of (125)I- and (18)F-labeled oligonucleotides are reported.     

1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins

FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.     

Theoretical CD spectrum calculations of the crown-ether aralkyl-ammonium salt complex

Rotatory strengths of the alpha-(1-naphtyl)-ethylammonium perchlorate (NEA)-phenazino-18-crown-6 ether molecular complex is determined theoretically by the coupled oscillator model and using ab initio random phase approximation (RPA) to describe local excitations on the chromophores. The computational results are compared to the experimental circular dichroism (CD) spectrum published previously. The good qualitative agreement between calculated and measured optical rotatory strengths allows one to assign the CD bands of the complex in a unique manner.     

Spontaneous mutational variation for body size in Caenorhabditis elegans

We measured the impact of new mutations on genetic variation for body size in two independent sets of C. elegans spontaneous mutation-accumulation (MA) lines, derived from the N2 strain, that had been maintained by selfing for 60 or 152 generations. The two sets of lines gave broadly consistent results. The change of among-line genetic variation between cryopreserved controls and the MA lines implied that broad sense heritability increased by 0.4% per generation. Overall, MA reduced mean body size by approximately 0.1% per generation. The genome-wide rate for mutations with detectable effects on size was estimated to be approximately 0.0025 per haploid genome per generation, and their mean effects were approximately 20%. The proportion of mutations that increase body size was estimated by maximum likelihood to be no more than 20%, suggesting that the amount of mutational variation available for selection for increased size could be quite small. This hypothesis was supported by an artificial selection experiment on adult body size, started from a single highly inbred N2 individual. We observed a strongly asymmetrical response to selection of a magnitude consistent with the input of mutational variance observed in the MA experiment.     

Marker chromosomes associated with tumour growth potential in plants

Abstract. The tumour growth potential of single-cell clones derived from the habituated tobacco strain Tabac anergié was analysed by: (1) culture on a medium which prevents the growth of normal cells, (2) graft tests, and (3) detailed chromosome analyses. Basal medium (BM) was more suitable for screening tumour cells than the hormone-free medium of Murashige and Skoog. Experiments using BM have pointed to the existence of different degrees of tumour growth potential. This is also indicated by graft tests which show variations of tumour growth potential at the intractional and interclonal levels. The chromosome analyses show a lack of correlation between chromosome number and tumour growth potential, but a good correlation between the latter and the number of marker chromosomes specific to tumour cells: the least-square regression line (y=13.76x+4.4) shows that the size of tumours is proportional to the number of marker chromosomes per cell. Moreover, the transition from a weak tumour state to a high tumour state by screening the tumour cells containing marker chromosomes on BM, reinforces the relationship between marker chromosomes and tumour development. These findings are relevant to the problem of the transformation of plant tissues, either by chromosome translocation, as is the case in many malignant cells, or with the exogenous T-DNA of the plasmid Ti carried by the bacterium Agrobacterium tumefaciens.     

The ras-related ral gene maps to chromosome 7p15-22

Summary Human cDNAs coding for the new protein ral that shares 50% homology with the ras proteins have been recently isolated. A 600-bp fragment carrying mainly the coding region was used to localize the ral gene by hybridization with sorted chromosomes and in situ hybridization. Direct molecular hybridization on sorted chromosomes using a single laser illumination allowed the assignment of the ral gene to a region of the flow karyotype containing chromosomes 7, 8 and X. With dual laser analysis ral was assigned to the fraction containing chromosome 7. In the 331 human metaphases hybridized with the ³H-labelled insert, the silver grain distribution showed a unique major signal on chromosome 7p15-22.     

Induction of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium by a barbiturate analog, 1-[2-phenylbutyryl]-3-methylurea

Summary In previous publications from our laboratory, we reported that a soluble, cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 can be induced by phenobarbital and a variety of other barbiturates. The tested barbiturates showed an excellent correlation between increasing lipophilicity and increasing inducer potency (Kim BH, Fulco AJ; Biochem Biophys Res Commun 116: 843–850, 1983). The only exception proved to be mephobarbital (N-methylphenobarbital) which, although more lipophilic than phenobarbital, is not an inducer of fatty acid monooxygenase activity. We have now found that 1-[2-phenylbutyryl]-3-methylurea (PBMU), an acylurea that can be derived from mephobarbital by hydrolytic cleavage of the barbiturate ring, is an excellent inducer of this activity. Paradoxically, the addition of mephobarbital to the bacterial growth medium containing PBMU significantly enhances the apparent potency of the acylurea to induce fatty acid monooxygenase activity as measured in cell-free extracts. When cell-free extracts of cells grown separately in PBMU or mephobarbital are mixed no enhancement of activity is seen. This finding suggests that the effect of mephobarbital is to somehow increase the efficiency of PBMU as an inducer of the P-450-dependent fatty acid monooxygenase rather than to induce an activator of this enzyme or a rate-limiting component of the monooxygenase system. Finally, both mephobarbital and PBMU induce the synthesis of total cytochrome P-450 in B. megaterium although PBMU is a much more potent P-450 inducer. For cytochrome P-450 induction, however, there is no synergistic or even additive effect when mephobarbital and PBMU are used together in the bacterial growth medium.Abbreviations PBMU 1-[2-phenylbutyryl]-3-methylurea - M.P. melting point     

Trace metal levels in commercially prepared tissue culture media

Four trace elements, lead, copper, tin and zinc, in addition to certain electrolytes, were measured in 11 commercially prepared tissue culture media. Glass media bottles and plastic tissue culture dishes and flasks were treated with a HCl acid solution to determine the amounts of trace metals leached from their surfaces. Zinc, lead and copper were detected in all media. Tin was detected only in RPMI Medium 1640, fetal bovine serum, minimum essential medium and penicillin-streptomycin. It is possible that a major cause of variability in tissue culture experimental results may be due to effects on growth caused by fluctuation in trace element contamination from batch to batch. Variability in establishing primary cultures of corneal endothelial cells was traced to high lead levels in commercially prepared tissue culture media. A strong case is made for continued diligent efforts to expand analytical horizons and our definition of substances in culture media.     

Excretion of urea and ammonia and its effect on the acid-base balance of water in the habitat of the anuran amphibian Xenopus laevis. Action of metabolic alkalosis]

We studied in Xenopus laevis the effect of changing the salinity and the acid-base status of the ambient water on the total nitrogen catabolism and the nature of the nitrogen end products, urea and ammonia. Increase of the ambient osmolarity by addition of NaCl led to a rise in protein catabolism and to a predominant ureotelism which can approach 95% of the excreted nitrogen. The osmolarity can reach 500 mosmol. L-1 without obvious harmful effects. NaCl can then be replaced by NaHCO3 without injury to the animal as long as water alkalosis is avoided by an appropriate increase of the ambient CO2 tension, PCO2. However, if PCO2 is kept low, the resulting water metabolic alkalosis causes death within a few hours.