Development and Localization of the Thymidine Phosphorylating Systems in Brain

Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[³H]thymidine at 37°C under 95% O₂-5% CO₂. When slices from all brain regions of 2-day-old rabbits were incubated in [³H]thymidine for 30 min, tissue-to-medium ratios of ³H were between 2 and 4 and declined with age, and the percentages of the total ³H in perchloric acid homogenates of brain slices as [³H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the ³H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [³H]thymidine for 30 min, the highest percentage of [³H]thymidine phosphates plus [³H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [³H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [³H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span.     

The proliferative response of rat liver parenchymal cells after partial hepatectomy A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine

Abstract. DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [³H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [³H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination.
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [³H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [³H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G¹-fractions was only 2 and 7%, respectively.     

Metabolism of Deoxyuridine in Rabbit Brain

Abstract: The metabolism of [³H]deoxyuridine by rabbit brain was investigated in vitro and in vivo . In vitro , brain slices from various regions of brain and from all age groups accumulated [³H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [³H]deoxyuridine was metabolized to [³H]deoxyuridine phosphate, with subsequent conversion to [³H]thymidine phosphate, and ultimately [³H]DNA. The percentage of the [³H]deoxyuridine phosphorylated and subsequently converted into [³H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [³H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [³H]deoxyuridine to [³H]thymidine phosphate in brain slices. In vivo , 2.5 h after intraventricular injection of [³H]deoxyuridine, over 90% of the [³H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the ³H remaining in brain was phosphorylated and converted to [³H]thymidine phosphates; and 11 and 4%, respectively, of the ³H remaining in brain was converted to [³H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life.     

Rate of Incorporation of R3hlthymidine In Various Tissues of the Mouse A basis for the evaluation of genotoxic effects of chemicals

Abstract. [³H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [³H]thymidine in vivo. [6-³H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [³H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [³H]thymidine incorporation into DNA.     

EFFECT OF PENTOBARBITAL ON MITOCHONDRIAL SYNTHESIS IN CULTURED GLIAL CELLS

Abstract— Primary cultures of glial cells prepared from brains of newborn rats were grown for 1 week and then exposed to 5 × 10⁻⁴ m -pentobarbital (PB) for 4 weeks. Compared with glial cells grown in drug-free medium throughout, exposure to PB significantly increased hexokinase activity, primarily in its mitochondrial form. Furthermore, cellular protein and RNA concentrations were significantly higher in barbiturate-cultivated than in control cells. Pulse labelling with [³H]thymidine after 4 weeks of PB exposure resulted in a significant increase (332%) in ³H incorporation into mitochondrial DNA while ³H incorporation into nuclear DNA was reduced by 58%. In addition, there was a time dependent increase in the size and number of mitochondria as determined in electron micrographs. These results are interpreted to reflect an increased mitochondrial metabolism in glial cells after chronic exposure to the barbiturate and may constitute a compensatory mechanism to the depressant action of this drug.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Acinar cell proliferation in the parotid and submandibular salivary glands of the neonatal rat

Abstract. Although the rat salivary glands are deficient in acini at birth, acinar cells proliferate rapidly during the early post-natal period. The pattern of acinar cell proliferation was analysed in the parotid and submandibular glands of neonatal rats from day of birth until day 34. Mitotic and [³H]thymidine ([³H]TdR) labelling indices of the two glands show distinctly different patterns. Analysis of cell division in the rat parotid gland demonstrated a peak of mitotic index at 14 days (2.9 ± 0.4%) and labelling index at 16 days (25.2 ± 2.1%). Submandibular gland acinar cell proliferation reaches a zenith between 7–8 days; labelling index (14.2 ± 1.1%) and mitotic index (2.3 ± 0.3%). Cell proliferation decreases rapidly in both glands after reaching a peak in activity. Gland size increases more rapidly in the submandibular gland which correlates with the earlier shift from cell proliferation to differentiation which occurs in this organ. Circadian rhythms of [³H]TdR incorporation were also investigated in this study. A circadian rhythm of [³H]TdR incorporation into DNA occurs at 15 days after birth with a peak at 06.00 hours in both glands and a trough occurring at 15.00 hours in parotid gland and 18.00 hours in the submandibular gland. Determination of specific activity of DNA (ct/min per μg DNA) on days 8, 10, 12, 13, 14, 15, and 16 after birth at 06.00 and 15.00 hours indicated that a circadian rhythm in [³H]TdR incorporation into DNA began on day 14. The developmental switch from suckling to solid food may be an initiating factor in the sychronization of the circadian rhythm in cell proliferation.     

Action of cortisol on the proliferation of rainbow trout fibroblasts

Abstract The effect of cortisol on the proliferation of the rainbow trout fibroblast cell line, RTG-2, was examined in synchronous and asynchronous cultures. When the transition from G₁ to S was synchronized by restoring serum to serum-deprived cultures, the addition of cortisol at the time of serum restoration delayed the entry of cells into S phase. However, if cortisol was added 24 h after serum restoration, at the G₁/S transition point, the subsequent peak of DNA synthesis was unaffected. In asynchronous cultures cortisol inhibited [³H]-thymidine and [³H]-uridine but not [³H]-leucine incorporation into acid-insoluble material. If the exogenous nucleoside concentration was raised, [³H]-thymidine but not [³H]-uridine incorporation continued to be inhibited by cortisol. This suggested that cortisol's effect on [³H]-thymidine incorporation reflected a change in entry into S phase and not just on thymidine uptake and metabolism. Cortisol inhibited the proliferation of RTG-2 in asynchronous cultures. At 1000 ng/ml of cortisol a reduction in cell number became apparent before the RTG-2 cultures were confluent, whereas at 100 ng/ml the reduction only became evident in confluent cultures. The synthetic antiglucocorticoid, RU 486, which acts at the level of the corticosteroid receptor, blocked the growth inhibition by cortisol. These results suggest that cortisol regulates rainbow trout fibroblast proliferation via the corticosteroid receptor and that the G₁/S transition is one point at which this regulation occurs.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.     

Cell Position Dependence of Labelling Thymidine Nucleotides Using the De Novo and Salvage Pathways In the Crypt of Small Intestine

Abstract. About twice as much tritiated thymidine ([³H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([³H]UdR) is even throughout the crypt.
Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway.
Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [³H]UdR, but has no effect on [³H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [³H]TdR into DNA. the increased efficiency of thymidine utilization by crypt base cells is not attributable to (i) differences in accessibility of thymidine; (ii) differences in the rate of DNA synthesis or (iii) the size of the nuclei.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Localization and Mechanism of Thymidine Transport in the Central Nervous System

Abstract: The localization and mechanism of thymidine and deoxyuridine transport in the central nervous system were studied in vivo and in vitro . Previous studies have shown that thymidine enters brain from blood in part via the CSF. In vitro , isolated adult bovine cerebral microvessels, which readily concentrated and phosphorylated deoxyglucose, were unable to concentrate thymidine and deoxyuridine. In vivo , [³H]thymidine (0.2 μ M ) and [³H]deoxyuridine(0.4 μ M ) were not extracted more readily than [¹⁴C]sucrose in a single pass through the cerebral circulation of rats. In vivo , [³H]thyrnidine retention in CSF and brain after entry from blood was increased when the efflux of [³H]thymidine from CSF and the phosphorylation of [³H]thymidine in brain were depressed by the intraventricular injection of unlabeled thymidine. These studies and previous work suggest that the transfer of thymidine (and deoxyuridine) through the blood-brain barrier in either direction must be extremely low. The present studies are consistent with the postulate that thymidine is transported by an active transport system in the choroid plexus that transfers thymidine from blood into the CSF; from the CSF, the thymidine enters brain cells and is phosphorylated.     

Fatty acid composition and microbial activity of benthic marine sediment from McMurdo Sound, Antarctica

Abstract Signature lipids from the phospholipid esterlinked fatty acids (PELFA) of cell membranes were used to describe benthic microbial communities of 4 Antarctic sediments. Metabolic activities of the communities were determined by incorporation of [³H]thymidine into bacterial DNA and sodium [¹⁴C]acetate into membrane lipids. Biomass measurements from extractable phospholipid fatty acids per g dry wt. ranged between 6 to 76 nmol, or when converted to number of bacteria, 3.7 × 10⁸ to 4.5 × 10⁹ cells per g dry wt. The West Sound site at New Harbor contained the lowest biomass, while Cape Evans on the East Sound contained the greatest. A marked difference was also noted between sites in their sediment microbial community structure. The East Sound sites at Cape Armitage and Cape Evans contained a greater abundance of diatom marker lipids, whilst both sides of the Sound contained approximately the same relative amounts of bacterial groups distinguished using PELFA. Activity of sediment microorganisms measured by radiolabel incorporation under ambient conditions followed the trends of the biomass measurements. The East Sound sites were more active by an average of 45–73% for [³H]thymidine and possibly also for sodium [¹⁴C]acetate.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

Proliferation of cells undergoing chondrogenesis in vitro

Abstract. Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [³H] thymidine ([³H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [³H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [³H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [³H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype.
We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage.     

K-Opioid Agonist Modulation of [3H]Thymidine Incorporation into DNA: Evidence for the Involvement of Pertussis Toxin-Sensitive G Protein-Coupled Phosphoinositide Turnover

Abstract: A body of evidence has indicated that μ-opioid agonists can inhibit DNA synthesis in developing brain. We now report that K -selective opioid agonists (U69593 and U50488) modulate [³H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K -selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3,5-fold increase was evident. Cell labeling by [³H]thymidine was also inhibited by the K -opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbin-altorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.     

NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.