Mitotic activity of pial microvascular endothelium rat in postnatal ontogenesis

Mitotic activity of endothelium in pial microvessels has been studied with the aid of intra-vessel autoradiography in 4-, 12- and 30-day old rats using these parameters: number of labeled nuclei per vessel or per 100 mcm of its length, or per 1 mm2 endothelial surface area, as well as number of vessels with labeled endothelium cells. The first parameter was independent of vessel diameter. The other parameters had highest value in the pericapillary vessels in all rats. These values decrease with rat age. The highest rate of brain growth was revealed after reaching the greatest mitotic endothelium activity in pial bed.     

DNA synthesis in the Purkinje neurons

(3H) Thymidine is incorporated into some cerebellar Purkinje cells of 6- to 30-day-old rats. The frequency of labelled neuronal nuclei was higher in the 12- to 30-day old rats than in the 6- to 10-day-old animals. The grain distribution pattern in autoradiographs was mostly nucleolar amounting to three to ten grains. Some other local labels were revealed, too. Only six Purkinje cells among 42,000 studied in 21 rats possessed heavy label (25 to 50 grains) distributed throughout the nucleus. Control estimations with deoxyribonuclease, hot perchloric acid and covering the autoradiographs again established that the Purkinje cells synthesize DNA perhaps for the purpose of DNA surplus accumulation and/or DNA repair in the neurons.     

Mitotic activity of the endothelium of different-diameter blood microvessels in the mesentery of Wistar rats

The technique of intravascular autoradiography of 3H-thymidine labeled nuclei of microvessels endothelia was developed. The density difference of labeled cells in microvascular bed of mesentery was estimated. The maximal density of labelled nuclei was found in pre- and postcapillary vessels, and minimal--in capillaries and large arteries and veins of mesenteric bed. The number of labeled nuclei per vessel was found to be relatively constant. The density of labeled cells in 4-months old rats is less than that in 3 weeks-old ones.     

Effects of a 4-aminopyridine in calcium movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex

Abstract: The processes of acetylation, phosphorylation, and methylation of nuclear proteins in cerebral hemispheres of 10- and 30-day-old rats were investigated. The experiments were carried out in vitro by measuring the incorporation of labeled precursors into histones and nonhistone chromosomal proteins (NHP) extracted from nuclei and separated by polyacrylamide gel electrophoresis. The results obtained indicate that there are age-specific differences in the processes of phosphorylation and methylation of chromosomal proteins, whereas the acetylation process did not change significantly between 10 and 30 days of age. Electrophoretic analysis of histones indicated that the histone H₃ was labeled to a greater degree than the other fractions and showed major changes in the processes of phosphorylation and methylation during postnatal development. The electrophoretic analysis of NHP showed considerable changes between 10 and 30 days of age. Certain components of NHP became increasingly evident as the brain developed. The methylation of an as yet unidentified protein with a molecular weight of approximately 118,000 daltons occurred at both ages.     

Changes in the reactivity of microvessels of the pia mater system of rats during ontogenesis

Changes in microvascular reactivity in pia mater system have been studied under conditions of bilateral occlusion of common carotid arteries in rats of different age groups. The studies were performed on the 7th, 30th, 45th and 90th day of the postnatal development. The occlusion of common carotid arteries lasted 6 min in week-old animals and 9 min in rats of other age groups. The phase character of microvascular reactivity has been determined. It has been found that dilation of arterioles depends on the age of the animal. So dilation of precortical arterioles averaged 20-25% of the initial level in 7-day-old animals and increased to 60-75% in 30- and 45-day-old animals. It has been determined that the smaller is the diameter of the vessels the more expressed is their reactivity. It has been shown that a steady reaction of compensatory adaptation in microvessels in characteristic only of adult animals.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

Chromatin phospholipid changes during rat liver development

The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0.47-0.59 per cent of total chromatin in newborn animals and 0.22 per cent in 45-day-old animals. No such age-related differences are observed in the nuclei. The phospholipid composition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine.     

Age-dependent variation of rates of polyadenosine-diphosphoribose synthesis by cardiocyte nuclei and the lack of correlation of enzymatic activity with macromolecular size distribution of DNA

Cardiocyte nuclei from neonate (5-day-old) and from adult (90-day-old) male Wistar rats were selectively isolated by a technique that eliminates interference by noncardiocyte nuclei (Jackowski, G., and Liew, C. C. (1980) Biochem. J. 188, 363-373). Polyadenosine-diphosphoribose synthetase activity of cardiocyte nuclei of neonates was ten times greater than in nuclei of adult rats, as calculated from initial velocity linear rate measurements. The molecular size of DNA extracted from cardiocyte nuclei of neonates was significantly larger (peak at 54 S) than DNA of cardiocyte nuclei of adults (peak at 33 S) as determined by alkaline sucrose density gradient ultracentrifugation (Knopf, K. W., and Weissbach, A. (1977) Biochemistry 16, 3190-3194). Comparison of the molecular sizes of DNA extracted from whole cardiac tissue with DNA isolated from cardiocyte nuclei shows that no DNA fragmentation takes place during the process of isolation of nuclei. Polyadenosine-diphosphoribose glycohydrolase activity was about 30% higher in cardiocyte nuclei of neonates than in adults, but the activity represents only 10% of the rate of the synthetase. Results demonstrate that polyadenosine-diphosphoribose metabolism in differentiated tissues is not directly related to the molecular size of DNA.     

Lithium/pilocarpine status epilepticus-induced neuropathology of piriform cortex and adjoining structures in rats is age-dependent

Distribution of LiCl/pilocarpine status epilepticus-induced neuronal damage was studied in the piriform cortex and in adjoining structures in 12-day-old, 25-day-old and adult rats. No distinct structural and neuronal alterations were detected in the basal telencephalon in 12-day-old rats surviving status epilepticus (SE) for one week or two months. In 25-day-old rats a decrease in Nissl staining was evident. There was also cell loss and gliosis in the caudal 2/3 of the piriform cortex, in the superficial amygdaloid nuclei, in the dorsal and ventral endopiriform nucleus and in the rostrolateral part of the entorhinal cortical area. In adult animals, the topography of neuropathological changes in the basal telencephalon was comparable to those in 25-day-old rats. The damage in the caudal 2/3 or caudal half of the piriform cortex in adult rats with survival times one week or two months was characterized by a marked loss of neurons and striking glial infiltration. The thickness of the piriform cortex and superficial amygdaloid nuclei was significantly reduced. In 25-day-old and in adult animals the sublayer IIb and layer III of the piriform cortex was more affected, while sublayer IIa was less damaged. Parvalbumin (PV) immunocytochemistry revealed a significant decrease in the number of PV-immunoreactive neurons in the rostral piriform cortex and in the dorsal claustrum in animals surviving for two months.     

Onset of compensatory hypertrophy of interstitial tissue and Leydig cells in rats hemicastrated around the time of puberty

When rats were unilaterally castrated at 20, 30, and 40 days of age, only those rats hemicastrated at 40 days showed compensatory hypertrophy of the interstitial tissue and Leydig cells when killed 30 days after hemicastration. At the time of death, volume densities of interstitial tissue, Leydig cells, and vascular components were greater in 70-day-old hemicastrated rats than in intact rats of the same age. The total number of Leydig cells per testis in hemicastrated and intact rats was always the same at any age. Estimated Leydig cell volume in 70-day-old rats was twice that in intact rats. By contrast, the testes of 50- and 60-day-old rats at the time of death displayed essentially the same morphological features, regardless of whether animals were hemicastrated. The concentration of plasma testosterone was higher in 50-day-old controls than in hemicastrated rats. Seventy-day-old hemicastrated rats showed higher levels of plasma testosterone than controls. The level of plasma dihydrotestosterone in 60- and 70-day-old hemicastrated rats exceeded that in the controls. A significant increase in follicle-stimulating hormone was noted in 50- and 70-day-old hemicastrated rats compared to normal rats, while levels of luteinizing hormone were basically the same. The increase in Leydig cell volume, interstitial tissue volume, vascular component volume, and plasma testosterone level caused by hemicastration at 40 days of age differed from that at 20 and 30 days of age.     

Polyacrylamide gel electrophoresis of rat brain acetylcholinesterase: isoenzymes of normal rat brain

Abstract— Triton-solubilized acetylcholinesterase (EC 3.1.1.7) of rat brain was submitted to vertical flatbed polyacrylamide gel electrophoresis. Three anodally migrating isoenzyme zones with low relative mobilities could be resolved, each of which on quantitative densitometry appeared to consist of more than one subzone. More than 50 per cent of the total AChE activity was exhibited by the isoenzyme zone closest to the origin (isoenzyme zone 3). Regional differences in AChE isoenzyme activity were quantitative only with the caudate-putamen complex, midbrain, pons and medulla oblongata exhibiting relatively high content of the three isoenzymes and the cerebral cortex and olfactory bulb possessing weak isoenzyme activities. Intermediate levels of isoenzyme activities were observed in the cerebellum and hippocampus. In all areas examined, the relative percentage values for each isoenzyme remained constant. AChE isoenzymes from the forebrain, brain stem and cerebellum of 15- and 30-day-old rats appeared to have identical patterns. In brain stem, no quantitative differences could be detected in the isoenzyme activities between 15 and 30 days of age. At both ages, the isoenzymes of male and female rats did not show any qualitative differences. The single cholinesterase (EC 3.1.1.8) isoenzyme which could be identified in brain stem supernatants of 30-day-old rats was weakly reactive and appeared to have the same relative mobility as the major acetylcholinesterase zone, zone 3. Acetylcholinesterase isoenzymes failed to demonstrate any differential response toward varying concentrations of inhibitors and to changes in pH. While there were basic similarities in the acetylcholinesterase and cholinesterase isoenzyme patterns of brain, serum, liver, skeletal muscle and intestine, brain alone exhibited a marked preponderance of the acetylcholinesterase isoenzyme zone 3.     

Ontogeny of alpha-amylase circadian rhythms in rat parotid gland

The content of alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) and total soluble proteins of parotid glands (from rats exposed to a photoperiod of 14 hr light: 10 hr dark), have been determined every 2 or 3 hr over 24 hr periods in 15, 25 and 90-day-old rats. In 35-, 45- and 72-day-old rats, determinations were performed only at 0100 and 1400 hr. The alpha-amylase and total soluble protein contents from 90-day-old rats show a circadian variation, with a maximum value at 2200 hr and a minimum at 1400 hr. Parotids from 15- and 25-day-old rats also show a circadian rhythm. The minimum value is recorded at 0100 hr and the maximum at 1400 hr. At day 35 and after, there is an inversion of the amylase rhythm. In immature rats, it appears that alpha-amylase and soluble protein are under the influence of another synchronizer, whose timing is independent of that imposed by mastication of solid food.     

CATIONIC COMPOSITION OF RAT CEREBRAL CORTEX SLICES. COMPARATIVE STUDY DURING DEVELOPMENT

Abstract—

• 1 Slices of cerebral cortex isolated from 20-day-old rats, when incubated in an appropriate oxygenated saline, show only a slight increase in weight (± 0.4 per cent of initial fresh wt.). At 30 days, under the same conditions, this increase in weight is 12.4 per cent. This is similar to the value observed in adult animals (13.6 per cent).
• 2 Modifications of the cationic content of slices in the case of 20-day-old animals are very slight while in 30-day-old animals they are greater and similar to those observed with adults.
• 3 The inulin space of 20- and 30-day-old rats are the same. The swelling exhibited by the slices isolated from 30-day-old animals may reasonably be attributed to a cellular uptake of extracellular fluid. This is accompanied by an increase of the Naf concentration and by a decrease of the K⁺ concentration. The values observed are similar to those observed with adult rats.
• 4 Comparing our results with previous histological and histochemical data, it may be suggested that the modifications of cationic content, as well as the swelling of slices isolated from 30-day-old rats or adult animals, are solely attributable to changes affecting glial cells and more specifically the astrocytes.

     

Multiple forms of mutarotases from the kidney, liver, and small intestine of rats: purification, properties, subcellular localization and developmental changes

Comparative studies of mutarotase [aldose 1-epimerase, EC 5.1.3.3] from the kidney, liver and small intestine of rats were performed placing in the focus on the study of multiple forms. The findings obtained are as follows. Mutarotases from the kidney and liver of adult rats were both separated into four forms (types I-IV) by DEAE-cellulose column chromatography, whereas only two forms (types I and II) were detected in the small intestine. Liver mutarotase type I was further separated into types I1 and I2 by column chromatography on hydroxylapatite. Types I and II from the kidney and type II from the liver were purified to homogeneity as judged by isoelectric focusing on thin layer polyacrylamide gel. Of various physicochemical properties, only the Km for alpha-D-xylose and the isoelectric point were different among the multiple forms. Liver mutarotase was immunohistochemically localized in the nuclei of parenchymal cells and small intestine enzyme in the nuclei of mucosal cells, indicating similarity with the localization of kidney enzyme (in the nuclei of epithelial cells of renal tubules and glomeruli) which was reported in our previous paper [Experientia (1979) 35, 1094-1097]. The kidney mutarotase level increased gradually after birth and reached a maximum near adult level within 20 days. This developmental pattern was essentially the same as that in the liver but clearly different from that in the small intestine, in which the mutarotase activity of suckling rats was several times higher than that of adult rats. Distribution patterns of multiple forms (types I-IV) of the enzyme in the kidney and liver of 10-day-old rats were similar to those in respective tissues of adult rats. On the other hand, the small intestine of 10-day-old rats contained four forms (types I-IV), whereas there were only two forms (types I and II) in adult rats.     

Hormonal control of the development of tryptophan oxygenase in primary cultures of young rat hepatocytes

Developmental increase of tryptophan oxygenase (L--tryptophan: oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11) was studied using hepatocytes of neonatal rats in primary culture. Hepatocytes from rats of 2–30-days-old were isolated and cultured for 2 days. In cultured hepatocytes of 2-day-old rats, tryptophan (2.5 mM), dexamethasone (1.10^?5 M) and glucagon (1.10^?7 M) did not cause the appearance of tryptophan oxygenase. But the enzyme activity became detectable, when heptocytes from 5-day-old rats were incubated wiht tryptophan, the oxygenase could be induced precociously by dexamethasone, but not by glucagon. The effect of glucagon was first seen 2 weeks after birth. However, in hepatocytes of 9-day-old rats glucagon stimulated formation of cyclic AMP and protein kinase activity (EC 2.7.1.37) and also induced tyrosine aminotransferase (EC 2.6.1.5). When heptocytes of 9-day-old rats were cultured for 4 days, their tryptophan oxygenase became inducible by glucagon. Insulin almost completely inhibited precocious appearance of the enzyme activity evoked by tryptophan plus dexamethasone in hepatocytes of 9-day-old rats. These results suggest that the appearance of tryptophan oxygenase in rat liver during development is due to first the onset of gene coding for tryptophan oxygenase and then stimulation by the sequential of glucocorticoid and glucagon.     

Proliferative processes in the myocardium in different parts of the heart during the creation of experimental myocardial infarct of the left ventricle in 2- and 3-week-old rats

As a result of 30 times repeated injections of 3H-thymidine (3HTdr) to neonate rats, beginning from days 13 or 21 post partum, ca. 20 and 10% of myonuclei in the left and right atria were labeled, respectively, while in both ventricles cumulative labeling of myocytes was nearly ten times lower. In rats of the same age with experimental infarction of the left ventricular myocardium the number of myonuclei labeled after 30-fold 3HTdr injections increased in atria up to 40-50%, in perinecrotic myofibers of the left ventricles up to 8-11%, and in myofibers of the left and right ventricle located far from the necrotic foci up to 3-4 and 2-3%, respectively. In some of rats subendocardial and/or subepicardial layers of the surviving left ventricular myocardium contained up to 15-35% of labeled myonuclei. Thus, in neonatal rats the extent of DNA synthesis reactivation in the nuclei of cardiomyocytes, the majority of which have recently completed normal ontogenetic proliferation, is, on the whole, of the same order as found in similar experiments on adult rats (Rumiantsev, Kassem, 1976; Oberpriller et al., 1984). However, still immature ventricular myocytes of neonatal rats resume mitotic cycle easier than those of adult animals which is evidenced not only by higher numbers of 3HTdr labeled myonuclei in subepicardial and subendocardial ventricular myocardia of some rats, but even more by reactivation of DNA synthesis in a limited fraction (2-3%) of the whole population of non-perinecrotic myocytes in both ventricles. Besides, reactive proliferation of cardiomyocytes in the atria of neonate rats, unlike in adults, starts on day 3 rather than on day 5 after infarction is induced. In the atria of neonatal rats polyploidization of myonuclei at later postinfarction stages is less pronounced than in adult rats which may be accounted for by formation of individual daughter nuclei during acytokinetic mitoses or, more seldom, by completion of cytotomy.     

Replenishment and nuclear retention of oestradiol-17 beta receptors in rat uteri during postnatal development.

1. Uteri of 6--10-day-old rats do not show a late growth response to oestrogen (increase in rate of DNA synthesis and cell division) exhibited by fully competent (20 days or older) uteri. We posed the question whether the lack of the late growth response is due to an inability to replenish the cytoplasmic pool of oestrogen receptors or to curtailed retention of oestrogen binding in the nucleus. Uterine nuclear and cytoplasmic receptors were measured by a [3H]oestradiol-17 beta exchange assay, at 1, 3, 6, 14 and 24 h after oestrogen injection. 2. The replenishment of cytoplasmic oestrogen receptors showed a similar pattern in the uteri of 6 and 10-day-old (partially responsive) and in 20-day-old (fully responsive) rats. 3. Oestrogen was retained longer in uterine nuclei obtained from 5 and 10-day-old rats than in uterine nuclei of 20 and 25-day-old rats. 4. Oestrogen receptors resistant to 0.4 M KCl extraction (residual receptors) were found in uterine nuclei of 6 and 25-day-old rats after oestrogen injection at all the times tested. The concentration of these residual receptors during the late period (6--24 h after injection) was not significantly different in uterine nuclei of 6-day-old and 25-day-old rats. 5. We conclude that neither lack of oestrogen receptor replenishment nor curtailed retention of oestrogen binding in the nucleus is the factor which limits the complete responsiveness to oestrogen in uteri of rats during postnatal development.     

The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from 10-, 30- and 60-day-old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase-bright rounded cells having diameters after fixation of 7-12 μm. The contamination was primarily by red blood cells and phase-dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pg/cell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals.     

An assay of RNA synthesis in hepatic nuclei from control and Streptococcus pneumoniae-infected rats

Hepatic nuclei were isolated, purified, and partially characterized from control and Streptococcus pneumoniae-infected rats. Biochemical and morphologic examination showed little contamination by other cell organelles. An in vitro system for the incorporation of 2-[14C]uridine-5'-triphosphate into ribonucleic acid (RNA) was developed and characterized. Although the stimulatory effects of cytosol on incorporation of labeled precursors into RNA have been previously reported, nuclei isolated from the livers of S. pneumoniae-infected rats were stimulated to a significantly greater extent than were nuclei isolated from the livers of control rats. In the presence of cytosol prepared from either control or infected rats, the increased incorporation of labeled precursor into RNA by nuclei isolated from infected rats was observed over broad pH and temperature ranges. The increased activity of infected nuclei was eliminated when albumin was substituted for cytosol, and could not be accounted for by differences in endogenous precursor pool size. These results are consistent with other infection-induced alterations in hepatic RNA and protein synthesis.     

REUTILIZATION OF LYMPHOCYTE DNA BY CELLS OF INTESTINAL CRYPTS AND REGENERATING LIVER

Lymphoid cells from mice injected 54 hours and 30 hours earlier with ³H-thymidine were washed and transfused into isogenic recipients at 29 to 30 hours after partial hepatectomy. The recipients were killed 28 to 30 hours later, and liver, intestine, Peyer''s patch, spleen, and the transfused cells were examined in autoradiographs exposed 6 months. Approximately 80 per cent of the labeled transfused cells were classed as lymphocytes. The labeled DNA contained in the transfused cells was partitioned to about 14 times as many recipient liver and intestinal cells, appearing in 72 to 78 per cent of hepatocyte nuclei, in 30 to 35 per cent of liver reticuloendothelial nuclei, and in 90 to 95 per cent of intestinal crypt nuclei. The label was not comparably widespread in the lymphoid organs, but was limited to a few intensely labeled lymphocytes and a somewhat larger number of very weakly labeled cells. When heat-killed cells rather than living cells were transfused, intensely labeled lymphocytes were absent from the lymphoid organs, but the labeling of cells in the recipients was otherwise identical. The results suggest that (a) reutilized DNA is derived from dead cells, (b) reutilized DNA is mainly degraded to nucleosides and nucleotides, the usual immediate de novo DNA precursors, before reincorporation into DNA, and (c) DNA reutilization may occur in the lymphoid organs, but on a less active scale than in intestine or regenerating liver.