Matrix area activity in the regenerating optic tectum of Rana esculenta

After keeping specimens of Rana esculenta at 4 degrees C for 24 hrs., the Authors removed a plug of right optic tectum. Thirty days later they injected 6-H3 thymidine and on the 200th day after surgery sacrificed the animals. With this technique it is possible to show the high regenerative capacity of the optic tectum; in fact, it is possible to observe reconstruction of the typical cellular and fibre layers of the removed nervous area. The origin of the cellular elements which supported this regenerative process and the possible connection between these results and restoration of the hemotrophic embryonic conditions due to absence of the blood-brain barrier, consequent on the cold treatment, is discussed.     

An autoradiographic study of the origin of intestinal blastemal cells in the newt, Notophthalmus viridescens.

Fifty adult newts were used in this investigation; in 44 animals, the intestine was transected perpendicular to its longitudinal axis approximately midway between pylorus and rectum. The free ends of the intestine were held in apposition with a single suture and replaced into the coelom. The animals were injected intraperitoneally with [³H]thymidine from 0 to 35 days after transection of the intestine and killed 6 hr later. In nontransected, control intestines, the only tissue that incorporated [³H]thymidine was the mucosal epithelium. In transected intestines, only the mucosal epithelium labeled in animals which had been injected with [³H]thymidine from 0 to 4 days after the intestine was incised. Later on, serosal cells and smooth muscle cells of the intestinal stump underwent morphological alteration, initiated the incorporation of [³H]thymidine into DNA, and began replication. At 6 days after transection, serosal cells adjacent to the plane of transection were incorporating [³H]thymidine and, at 12 days, smooth muscle cells at the transected surface were labeling. It seems probable that they both furnished cells to the intestinal blastema; the lining epithelium of the mucosa, however, did not appear to contribute to the blastema proper.     

Effect of 5-fluoro-2'-deoxyuridine on [h]thymidine incorporation by bacterioplankton in the waters of southwest Florida

The effect of 5-fluoro-2'-deoxyuridine (FdUrd) on [methyl-H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-H]thymidine or [6-H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [H]thymidine labeling of protein and RNA, but caused some inhibition of [H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [H]thymidine incorporation.     

Synergism between prolactin and ovarian hormones of DNA synthesis in rats mammary gland.

Autoradiography with [30H] thymedine has been used to measure the rate of DNA synthesis in the mammary epithelium of hypophysectomized-ovariectomized rats under the influence of estradiol, progesterone, and prolactin. Controls and animals treated with estradiol did not increase [3-H] thymidine incorporation, while progesterone alone had a slight stimulatory effect. Prolactin alone stimulated some [3-H] thymidine uptake in ductal and alveolar epithelium, but when combined with either estradiol or progesterone synergistic effects were observed. Estradiol with prolactin stimulated incorporation primarily in the ductal epithelium, whereas progesterone with prolactin stimulated both ductal and alveolar epithelium.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.     

In vivo and in vitro synthesis, release, and uptake of [3-H]-dopamine in mouse striatal slices after in vivo exposure to chlordecone

Effects of treatment of mice with chlordecone (25 mg/kg/d) on striatal dopaminergic activities such as synthesis, turnover, uptake, and release were investigated in vivo and in vitro. In mice receiving chlordecone for five days, there were no significant changes in in vivo dopamine (DA) synthesis and turnover in striatum and in vitro [3-H]-dopamine uptake and K+-stimulated [3-H]-dopamine release in striatal slices. In mice receiving chlordecone for eight days, the in vivo synthesis of [3-H]-dopamine from [3-H]-tyrosine in striatum was slightly inhibited and the in vitro [3-H]-dopamine synthesis in striatal slices was significantly decreased. Furthermore, both uptake and K+-stimulated release of [3-H]-dopamine from striatal slices were significantly reduced. The turnover rate of newly synthesized [3-H]-dopamine from [3-H]-tyrosine in striatal slices was unchanged after eight consecutive days of chlordecone administration. These results suggest that chlordecone may cause impairments in pre- and/or postsynaptic membranes of dopaminergic neurons which modulate motor function.     

THE EFFECT OF INTRAOCULAR INJECTION OF CORDYCEPIN ON RETINAL RNA SYNTHESIS AND ON RNA AXONALLY TRANSPORTED DURING REGENERATION OF THE OPTIC NERVES OF GOLDFISH

Abstract— The presence of relatively large amounts of RNA has been demonstrated in regenerating axons of the goldfish optic nerve. Previous experiments have suggested that this R NA may be composed of only small molecular weight 4S RNA. The present experiments were performed in order to see if inhibiting RNA transport by intraocular injections of cordycepin causes a selective depletion of 4S RNA arriving in the contralateral optic tectum, and thus add further evidence that 4S RNA is axonally transported. Optic nerves were crushed in a group of goldfish and 18 days later 10.0 /tg of cordycepin was injected into the right eye followed 3 h later by injections of [³H]uridine into the same eye. Six days later the amount of axonally transported [³H]RNA was decreased by 89% compared with non-cordycepin treated controls. The effect of cordycepin on retinal RNA synthesis was shown by autoradiography to be primarily on retinal ganglion cell RNA synthesis with lesser effects on other cellular elements of the retina. SDS polyacrylamide gel electrophoresis at both 1 and 6 days after intraocular injections of cordycepin and [³H]uridine, showed that cordycepin blocks the retinal synthesis of ribosomal RNAs but appeared to have little effect on the synthesis of 4S RNA. When transported RNA in the tectum was fractionated by gel electrophoresis 6 days after injection, it was found that the amount of ribosomal RNA was decreased by approx 70% as a result of cordycepin pretreatment. This correlated well with the effect of cordycepin on the transport of available RNA precursors (also decreased by approx 70%) and is consistent with the contention that in these experiments ribosomal RNA is synthesized in the tectum itself and is not axonal. The amount of [³H] 4S RNA arriving in the tectum, however, was decreased by greater than 90% suggesting that its presence in the tectum was not entirely dependent on the availability of ³H precursors for local synthesis in the tectum. These results are consistent with data suggesting that 4S RNA is the predominant, if not the only, RNA species axonally transported during regeneration of goldfish optic nerves.     

THE TURNOVER OF ISOTOPICALLY LABELLED DNA IN VIVO IN DEVELOPING, ADULT AND SCRAPIE-AFFECTED MOUSE BRAIN

Abstract— Tracer experiments using [³H]thymidine have shown that a large proportion of the DNA synthesized in control and scrapie-affected mouse brain is metabolically unstable. Although the turnover of mitochondrial DNA contributed to the loss of radioactivity from whole brain, it has been shown that 70 per cent of the labelled nuclear DNA was removed between 1 and 21 days after injecting the isotopic precursor. Observations on developing mouse brain, where the rate of DNA synthesis is far higher than that in adult brain, also revealed the existence of metabolically unstable DNA. Similar studies on developing and adult brain using [¹⁴C]thymidine indicated that most of the radioactivity lost in vivo was not due to radiation damage to newly labelled DNA molecules. Hydroxyapatite chromatography of heat denatured and renatured DNA from adult brain showed that the rates of turnover of the poorly, moderately and highly reiterated species of nuclear DNA were similar. The results of some dissection experiments have further shown that the observed breakdown of DNA in adult brain was not specifically associated with the turnover of subependymal cells. It is suggested that a metabolically labile fraction of nuclear DNA is present in developing and adult mouse brain and that the amount of tracer incorporated into this fraction is increased in mice infected with scrapie.     

AXONAL TRANSPORT OF RADIOACTIVITY IN THE GOLDFISH OPTIC SYSTEM FOLLOWING INTRAOCULAR INJECTION OF LABELLED RNA PRECURSORS

After injection of the tritiated RNA precursors [³H]guanosine, [³H]uridine or [³H]orotic acid into the eye of goldfish, labelled TCA-soluble material and RNA appeared to be axonally transported to the contralateral optic tectum. From the time courses of arrival in the tectum,‘average’rates of transport of 6 mm/day for the soluble material and 1·7 mm/day for the RNA were calculated. If the optic nerve was cut after the transported material had arrived in the tectum, about 60 per cent of the TCA-soluble material disappeared by 7 days after the cut, but almost none of the RNA. After a further 8- to 13-day period, the TCA-soluble material had declined by a further 50 per cent from the 7-day value, but the RNA by only 20 per cent. Thus, relatively little RNA was lost when the optic axons degenerated, an observation which suggested that the RNA might be extra-axonal. However, if the optic nerve was crushed before the arrival of the transported material, RNA did not appear in the tectum until the regenerating optic nerve fibres arrived. Therefore, the presence of RNA must be dependent on intact nerve fibres. Moreover, in the earliest stages of regeneration the proportion of transported RNA to TCA-soluble material was considerably higher than normal, suggesting that the regenerating fibres arrived in the tectum already carrying RNA. This implies that the RNA itself was transported in the optic fibres.     

Evaluation of radiation dose resulting from the ingestion of [3H]- and [14C]thymidine in the rat

Average doses to rat tissues from the ingestion of 2-[14C]thymidine were compared with those from methyl-[3H]thymidine or 6-[3H]thymidine. Among the three precursors, [14C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [14C]thymidine were almost the same or lower as compared with those from [3H]thymidine, irrespective of the 9 times higher beta-ray energy of 14C than that of 3H. In the case of [14C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [3H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (3HHO), formed by degradation of [3H]thymidine. No significant difference in their total doses was found between the two [3H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[3H]thymidine than with 6-[3H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [3H]thymidine were also made in order to predict more precisely possible radiation hazards.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Effects of chronic isoproterenol administration on beta 1-adrenoceptors and growth of pancreas of young and adult rats

[3H]Dihydroalprenolol (DHA) binding of membranes of adult pancreas differed from that of pancreas of young rats, and the DHA binding in the presence of atenolol or butoxamine also was different in the two age groups. The adult pancreas had 93% beta 2- and 7% beta 1-adrenoceptors and did not exhibit an increased incorporation of [3H]thymidine into deoxyribonucleic acid (DNA) following 2 days of DL-isoproterenol (ISO) administration; in contrast, pancreas of the 20-day-old rat had 71% beta 2-adrenoceptors and 27% beta 1-adrenoceptors and exhibited a 34-fold increase over that of adult, and a 6-fold increase over that of the control 20-day-old pancreas. Acinar cell differentiation was also accelerated by a 7-day regimen of ISO administration from 13 to 20 days of age. These growth responses to ISO appear to be beta 1 mediated. The lack of beta 1-adrenoceptors in the adult may account for the failure of the adult pancreas to exhibit a growth response to ISO.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

In vivo studies on the metabolism of pyrimidine nucleosides (author's transl)]

3H-labelled metabolites were determined in the perchloric acid-soluble fraction of blood plasma and liver of adult male Wistar rats, following the application of [5 - 3H]uridine. Ten minutes after the injection of uridine, only 20% of the total 3H activity of the plasma could be attributed to [3H]uridine. The remaining radioactivity was found chiefly in [3H]uracil (40%) and 3H2O (20%). In the liver, at 10 min, [3H]-uridine and [3H]uracil together accounted for less than 0.5% of the total radioactivity; about 70% of the radioactivity was due to [3H]beta-alanine, and 15% to 3H2O. 45 min after the injection, 70% of the radioactivity in the plasma was due to 3H2O, whereas uridine and uracil represented about 4% and 6%, respectively. At this time, about 55% of the radioactivity in the liver was due to [3H]beta-alanine, about 40% to 3H2O, and about 5% to unidentified metabolites; [3H]uridine and [3H]uracil were not observed. A comparison of the rate of catabolism of [5-3H]-uridine, [5-3H]cytidine and [6-3H]thymidine showed that cytidine is degraded in the organism 25 times more slowly than uridine or thymidine. The biological half lives for the total degradation of the [3H]nucleosides to 3H2O, based on the values in the plasma, were: uridine 1.1 h; thymidine 1.3 h; cytidine 25 h. Furthermore, the turnover time of exogenous uridine in the plasma was found to be 9 min, which gives a half life of 6 min for the metabolism of exogenous uridine to uracil.     

Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet.

The feeding of a high-fat diet to adult rats was shown to increase the incorporation of [3H]thymidine into DNA of the adipocyte and stromal fractions. After only 2 days on a high-fat diet there was a marked increase in the incorporation of label. When a 2-week period was interposed between [3H]thymidine administration and determination of DNA specific activity, the greatest increase in incorporation of label was found after 1 week on the diet, when incorporation increased 6-fold or more in both adipocytes and stroma and subsequently decreased to stabilize at a level two or three times that of chow-fed rats in the adipocyte fraction. Rats labeled when young and later placed on a high-fat diet showed a decrease in DNA specific activity in both adipocytes and stroma, confirming that cellular proliferation had occurred in both fractions. The specific activities of both stromal and adipocyte DNA were very similar at all time points studied. An attempt to increase the difference in specific activities by waiting many weeks after [3H]thymidine injection before isolating DNA was not successful. This may be because the total amount of DNA in the stromal and adipocyte fractions increases in parallel on the diet. The significance of these findings in terms of the normal turnover of adipose tissue DNA and the responsiveness to diet is discussed.     

Growth hormone inhibits renotropic action of luteinizing hormone-isoform(s)

We recently purified luteinizing hormone (LH)-isoforms with renotropic activity from ovine pituitaries based on the stimulation of [3H] thymidine incorporation into renal DNA of castrated-hypophysectomized rats. In this study, we examined the hormonal interactions between ovine growth hormone (GH) and this LH-isoform in renal DNA synthesis. A single injection of LH-isoform (40 micrograms) significantly increased [3H] thymidine incorporation, but an injection of GH (200 micrograms) did not, during experimental periods of up to 26 hours. Repetitive ovine GH treatment (5 days) did not change basal [3H] thymidine incorporation, either, although its biological activity was evidenced by an increase in insulin-like growth factor-I (IGF-I). Stimulated [3H] thymidine incorporation by LH-isoform (100 micrograms) was significantly suppressed by an injection of GH (200 micrograms) and was, to a greater extent, by repetitive treatment with GH (200 micrograms/day, for 3 or 5 consecutive days). These results demonstrated one example of the effect of complex hormonal interactions on kidney growth.     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

Organ culture of adult rat colonic mucosa on fibrin foam

A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37 degrees C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum, L-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmostphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland.     

Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.     

DNA synthesis in mouse brown adipose tissue is under beta-adrenergic control

The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of [3H]thymidine. Cold exposure led to a large increase in the rate of [3H]thymidine incorporation, reaching a maximum after 8 days, whereafter the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase [3H]thymidine incorporation. When norepinephrine was injected in combination with the alpha-adrenergic antagonist phentolamine or with the beta-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a beta-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.