Preferential Accumulation Of Radioactivity Research Articles

Monitoring Gene Therapy by External Imaging of mRNA: Pilot Study on Murine Erythropoietin

Gene therapy is anticipated as being an important medical development. Essential to its effectiveness is the appropriate activity (protein expression) in the expected target cells. A noninvasive diagnostic procedure of successful gene expression will be of paramount importance to validate its use or its misuse (eg, sports gene doping). Externally detectable labeled oligonucleotide hybridizing with the messenger RNA generated by the transferred gene has been proposed as a possibility to monitor successful gene therapy. The authors selected the erythropoietin gene (Epo) for a pilot study on erythropoietin protein expression in mouse muscle. Oligonucleotides of peptide nucleic acid (PNA) type capable of antisense binding to unique murine Epo-mRNA sequences were synthesized by solid phase methods, and elongated at the N-terminus with the HIV Tat (48-60) cell penetrating peptide. They were labeled with fluorescence and radioactive tags to verify penetration and longer half-life properties in Epo gene transfected C2C12 mouse muscle cells as compared with corresponding wild-type cells. Downregulation of newly expressed erythropoietin protein in such cells additionally confirmed the penetration and hybridizing properties of the selected labeled oligonucleotide. I-labeled Tat-PNAs were intravenously injected into mice that had previously received the Epo gene into the right tibialis muscle by DNA electrotransfer. Preferential accumulation of radioactivity in the transferred limb as compared with the contralateral limb was ascertained, especially for I-Tat-CTA CGT AGA CCA CT (labeled Tat-PNA 1). This study provides experimental data to support the potential use of external noninvasive image detection to monitor gene therapy. The extension of the approach to more sensitive methods for whole-body external detection such as positron emission tomography appears feasible.

Retrograde axonal transport following injection of [ 3H]serotonin in the olfactory bulb. I. Biochemical study

A retrograde axonal transport from the serotonergic nerve terminals in the olfactory bulb (OB) to their parent cell bodies in the midbrain raphe nuclei has been demonstrated after stereotaxic injection of [ 3H]5-HT into the OB of rats pretreated with a monoamine oxidase (MAO) inhibitor: at various time intervals thereafter (4–92 h) there was a preferential accumulation of radioactivity mainly in the raphe dorsalis nucleus (RDN). Maximal accumulation occurred at 24 h. Of this radioactivity, 30–50% was recovered as 5-HT. The accumulation was estimated to take place at two rates: a fast one (48 mm/day) and a slower one (16 mm/day). Under the same experimental conditions there was no clear evidence for a retrograde accumulation of [ 3H]norepinephrine in the RDN. A passive diffusion mechanism could be excluded since the diffusion of tracer towards the cerebrospinal fluid was prevented by prior mechanical obstruction of the olfactory diverticle of the lateral ventricle. Furthermore, colchicine strongly reduced (by 80%) the radioactive accumulation in the RDN. Destruction of serotonergic nerve terminals by 5,6-dihydroxytryptamine or inhibition of 5-HT uptake by fluoxetine decreased this retrograde accumulation whereas destruction of catecholaminergic nerve terminals by 6-hydroxydopamine was without effect. Pretreatment with reserpine decreased the amount of radioactivity transported to the RDN by 40%. In the absence of MAO inhibition pretreatment, animals still presented 35% of the tracer transported to the RDN. Intrabulbar injection of MAO inhibitor did not affect the accumulation rates when compared with animals which received the inhibitor by the intraperitoneal route.

Autoradiographic studies of 3H-estramustine in the rat ventral prostate.

The alkylating agent 3H-estramustine was administered to castrated male rats and found to accumulate in the epithelium of the ventral prostate 5 and 20 min. following intravenous administration, as judged from autoradiography. Four hrs. after intravenous injection of this isotope, the radioactivity was recovered in the secretion of the prostatic lobuli. A preferential accumulation of radioactivity in prostatic secretion was also observed 2 hrs. after intramuscular administration of 3H-estramustine to intact or castrated rats. In contrast, 3H-oestradiol and 3H-testosterone, which were also taken up by the ventral prostatic epithelium, were not recovered in prostatic secretion. The present results indicate that 3H-estramustine is secreted from the prostatic cells into the lumina of the prostatic lobuli. It is speculated that a recently detected estramustine-binding protein in rat ventral prostate may be involved in this process and that the present finding may be of relevance in the understanding of the mechanism of action of the chemotherapeutic agent Estracyt (estramustine phosphate) used in the treatment of advanced prostatic carcinoma.

Comparison between the retrograde axonal transport of nerve growth factor and tetanus toxin in motor, sensory and adrenergic neurons

In previous studies it has been shown that nerve growth factor (NGF) is taken up with high selectivity by adrenergic and sensory nerve terminals and is transported retrogradely to the corresponding cell bodies by a colchicine sensitive mechanism 10,11,23. The present study was designed to investigate whether this retrograde transport of NGF depends on properties of the nerve terminals which are common to all the neurons or restricted to those which respond to NGF either during the whole life cycle (adrenergic neurons) or during a restricted period of embryonic development (sensory neurons). In order to investigate the retrograde transport of NGF in motor neurons we injected [125I]NGF into the musculus deltoideus and measured the side differences of accumulation of radioactivity in the spinal cord (C6-C8) from 4-48 h. At no time was there a preferential accumulation of radioactivity on the injected side. In contrast, tetanus toxin was accumulated preferentially on the injected side and an approximate rate of transport of 7.5 mm/h was calculated. Astonishingly there was also a retrograde transport of tetanus toxin in sensory and adrenergic neurons. The rate of transport was identical to that of NGF and the transport could be blocked by transection of the corresponding nerve fibers and local administration of colchicine. After unilateral injection of [125I]tetanus toxin into the forepaw or the musculus deltoideus light microscopic autoradiographs revealed heavily labeled neuronal cell bodies in the dorsal root ganglia or the ventrolateral spinal cord of the injected side. It is concluded that the retrograde transport of [125I]NGF depends on properties of the neuronal membrane which are specific for adrenergic and sensory neurons, whereas that of tetanus toxin is determined by features which are common to all, or at least to all peripheral, neurons. The fact that the rate of transport for both NGF and tetanus toxin is identical in all examined neurons, supports the hypothesis that the specificity of retrograde transport is determined by specific uptake sites in the neuronal membrane whereas the retrograde transport system is non-specific.

Specificity of retrograde transport of nerve growth factor (NGF) in sensory neurons: A biochemical and morphological study

In previous studies it has been shown that nerve growth factor (NGF) is taken up with a high selectivity by adrenergic nerve terminals and is transported retrogradely to the perikaryon 11,22. It was the aim of the present experiments to investigate whether the sensory neurons exhibit the same high degree of selectivity for retrograde transport throughout the whole life cycle, although it is known that their dramatic response to NGF is confined to a short period of ontogenetic development. Unilateral injection of [ 125I]NGF into the forepaw of adult rats was followed by a preferential accumulation of radioactivity in the sensory ganglia (C 6–C 7) of the injected side. However, this preferential accumulation was not detectable earlier than 6 h after injection and reached a maximum (ratio between injected and non-injected side, 5:1) after 11–16 h. Transection of the plexus brachialis abolished and local administration of colchicine prior to that of [ 125I]NGF greatly reduced the preferential accumulation of radioactivity in the ganglia of the injected side. The rate of retrograde transport of NGF in sensory neurons was calculated to be 13 mm/h which is about 5 times faster than that in adrenergic neurons. The selectivity of this retrograde transport was demonstrated by the fact that injection of 125I-labeled bovine serum albumin and cytochrome c did not result in a preferential accumulation of radioactivity in the sensory ganglia of the injected side. Light microscopic autoradiography revealed heavily labeled cells in the sensory ganglia (C 6–C 7) of the injected side after administration of [ 125I]NGF into the forepaw. Only cells belonging to the large cell type were labeled. Prolonged (7 μg/g/day over 5 days) injection of NGF into the forepaw of 10-day-old rats did not result in a hypertropic response of the sensory neurons as far as can be judged from morphometric studies at the light microscopic level.

Uptake of anrogens by the brain of the neonatal female rat

Neonatal and juvenile female rats were given a subcutaneous injection of tritium-labeled testosterone propionate (TP) or testosterone (T), with or without added carrier. Levels of radioactivity in plasma, hypothalamus, amygdala and cerebral cortex were measured at various time intervals after injection. Free radioactive steroids in the brain were separated from conjugated steroids by solvent partition, and the free steroids were further fractionated by paper chromatography. (1) No preferential accumulation of radioactivity from plasma by the hypothalamus, amygdala or cortex was observed after injection of [ 3H]-TP or [ 3H]-T, irrespective of the age of the females (1, 5, 6, 11 or 21 days) and time of determination (1–48 h after injection): the ratio of brain region disint./min: plasma disint./min did not exceed 0.5. (2) Cold TP or T, administered simultaneously with [ 3H]-TP or [ 3H]-T, respectively, did not reduce the uptake of radioactivity by the hypothalamus, amygdala or cortex of the 5-day-old females. (3) The concentration of free steroids in the brain (disint./min/mg wet wt), 1–4 h after injection of [ 3H]-T, was several times higher than after [ 3H]-TP injection. At 8 h or later, the concentration of free steroids, and particularly of unconjugated testosterone 5α-dihydrotestosterone and androstenedione, was higher in the brains of the [ 3H]-TP-treated animals. (4) A decline in the uptake of [ 3H]-T was observed between the 1st and 6th days of life, with no further changes until the 11th day. Thus the progressive fall in effectiveness of TP in producing androgenization known to occur between the 6th to 11th days of life was not paralleled by changes in the uptake of [ 3H]-T by the hypothalamus, amygdala or cortex as assayed 2 h after injection. The results do not support the existence of a specific high-affinity low-capacity uptake mechanism for testosterone in the brain of the neonatal female rat comparable with the demonstrable in adrogen-responsive organs such as prostate gland.

Preferential Accumulation of Radioactivity in Root Nodules of a Legume after Dosage with 2,4,5‐T‐14C

Preferential Accumulation of Radioactivity in Root Nodules of a Legume after Dosage with 2,4,5‐T‐¹⁴C