The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

大鼠发育过程中肝、肺r-GT酶活性和定位

用生物化学和组织化学方法研究正常发育中大鼠肝、肺r-GT活性和定位。结果表明:肝r-GT活性自胚龄17天开始升高,21天达高峰,出生第一天明显降低,第六天降至接近成年低水平。在胚胎期肝r-GT主要位于肝细胞内,出生后则主要位于胆小管。该结果提示胚胎期肝r-GT主要参与肝细胞膜上氨基酸的转运。出生后可能主要参与解毒功能,大鼠肺r-GT活性随发育逐步升高,主要分布于肺支气管上皮细胞。提示肺r-GT可能参与解毒功能。     

Changes of Enzymes Involved in Prostaglandin Metabolism and Prostaglandin Binding Proteins in Rat Brain During Development and Aging

In the developing rat brain, the enzymatic formation of prostaglandin D2 from prostaglandin H2 increased 60-fold from day 12 of gestation to birth. The activity still rose gradually to the highest level (90 nmol/min/g wet tissue) at day 7 after birth. The activities of prostaglandin E2 and F2 alpha synthetases in rat brain were highest at gestational age 19 days (30 nmol/min/g wet tissue), respectively. The specific activity of NADP-dependent 15-hydroxy-prostaglandin D2 dehydrogenase in rat brain was highest at the earliest gestational age we examined (day 12 of gestation). The specific bindings of prostaglandin D2 and E2 to the crude mitochondrial fraction of rat brain were observed from day 16 of gestation and increased to day 7 after birth. Although the activities of the enzymes responsible for prostaglandin metabolism were unchanged postmaturationally, the maximal concentrations of the binding sites on the synaptic membrane for both prostaglandins D2 and E2 decreased with constant affinity to less than one-sixth with age from 1 week to 24 months after birth. These results indicate that prostaglandins may play important roles during maturation and aging in rat brain.     

Developmental Change in the Glycosaminoglycan Composition of the Rat Brain

Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre- and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (> 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age-related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.     

DNA synthesis and DNA polymerase activity in differentiating cardiac muscle

DNA synthesis in cardiac muscle of the rat declines rapidly following birth and is essentially “turned off” by the 17th day of postnatal development. Soluble DNA polymerase activity also decreases progressively with age, reaching adult levels of almost zero by the 17th day of development. These results indicate that cessation of DNA synthesis in differentiating cardiac muscle may be attributed to the loss or inactivation of DNA polymerase.     

A microtubule-associated protein (MAP1) which is expressed at elevated levels during development of the rat cerebellum.

Monoclonal antibody (MAb) G10 labels a single high mol. wt. (HMW) band on Western blots of microtubule preparations from 2 day old rat brain. The G10 antigen is thermolabile and co-migrates with microtubule-associated protein (MAP)1 from young rat brain on low percentage (5%) polyacrylamide-SDS gels. The G10 antigen decreases by about five times from birth to adulthood in the rat cerebellum. The same single band is labelled on Western blots of homogenates of whole neonatal rat brain but no labelling is found using neonatal or adult kidney, lung or liver. We have therefore identified a brain-specific MAP1, designated MAP1(x). Immunofluorescence microscopy using MAb G10 on parasagittal sections of rat cerebella shows labelling of the newly formed molecular layer in 6 day old rats. Only a narrow band close to the pial surface is labelled in 18 day old animals, which disappears in the adult. Labelling of the cerebellar white matter found in young rats also disappears. Neurones but not flat cells in cerebellar cultures label with MAb G10. All staining patterns are consistent with an axonal distribution of the antigen. MAP1(x) may be part of a developmentally regulated microtubule structure.     

Nicotinamide methylation tissue distribution,developmental and neoplastic changes

The distribution of cytosolic activity of nicotinamide:S-adenosylmethionine methyltransferase (nicotinamide methylase, EC 2.1.1.1) in normal tissues from adult rat and mouse and in tumors and the change in the enzyme activity during the the development of rat tissues were studied. (1) Rat liver exhibited the highest nicotinamide methylase activity among all adult tissues tested; other rat tissues, like adrenal, pancreas, kidney, brain and mouse tissues, had only less than 15% of the adult rat liver activity. (2) 3 days before birth, fetal liver showed a very low nicotinamide methylase activity (2% of adult rat liver), which, however, increased already 1 day before birth and reached the adult level on the day 28 after birth. (3) In a variety of hepatomas and ascites tumors, an inverse correlation, with some exceptions, between tumor growth rate and nicotinamide methylase activity could be seen. In all hepatomas, with the exception of Morris hepatoma 5123tc, nicotinamide methylase activity was significantly decreased in comparison to normal adult rat liver. The highly malignant Zajdela hepatoma, Yoshida sarcoma, sarcoma 180 and Ehrlich ascites tumor methylated nicotinamide only at a negligibly low rate. (4) Cultured RLC cells (an established rat liver cell line) from the stationary growth phase or G₁-arrested RLC cells had about half of the adult rat liver activity, yet the activity was 70% higher than that of the logarithmically growing RLC cells.     

Biochemical aspects of cardiac muscle differentiation. Deoxyribonucleic acid synthesis and nuclear and cytoplasmic deoxyribonucleic acid polymerase activity.

DNA synthesis and DNA polymerase activity have been measured in terminally differentiating cardiac muscle of the rat. Incorporation of [3H]thymidine into DNA essentially ceases by the 17th day of postnatal development. Cardiac muscle of neonatal rats contains at least two molecular species of DNA polymerase: a 3.5 S DNA polymerase that can be extracted from nuclei with 0.2 m potassium phosphate and a 6 to 8 S soluble cytoplasmic DNA polymerase. The nuclear DNA polymerase in crude extracts has a pH optimum of 9.0 and is more active with native DNA than with denatured DNA as the primer-template. The cytoplasmic DNA polymerase in crude extracts has a pH optimum of 7.5 and is more active with denatured DNA. The activity of the 6 to 8 S cytoplasmic DNA polymerase decreases 80-fold from day 1 to day 17 after birth, which correlates temporally with the reduced rate of DNA synthesis. The activity of the 3.5 S nuclear DNA polymerase remains relatively constant throughout postnatal development. Mixing experiments (assay of neonatal enzyme extracts with adult enzyme extracts) gave additive results, suggesting that the decline in 6 to 8 S DNA polymerase activity apparently is not due to the presence of absence of soluble activators or inhibitors at different times during development. These studies may provide a system which can be used to investigate the control of DNA synthesis and cellular proliferation during the terminal stages of cardiac muscle differentiation.     

Ontogenesis of Monoamine-Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration

Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co-factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands; the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12-day-old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co-factor, biopterin, in rat midbrain.     

Perinatal development of hepatic cholesterol synthesis in the rat

Rates of cholesterol synthesis and HMG CoA reductase activity in rat liver, have been reported to be high before and low after birth. The timing of the decline in perinatal rates of cholesterol synthesis, however, is uncertain. These studies, therefore, determined in vivo rates of cholesterol synthesis using [3H]water and hepatic reductase activity in vitro in perinatal rats. The lipid composition of the plasma, liver and its microsomal subfraction were also determined. Reductase activity increased during late gestation, remained high immediately after birth, then decreased with the commencement of suckling. Rates of cholesterol synthesis increased from gestation day 18 to 20, but in contrast to reductase activity, decreased on the day before birth. Plasma cholesterol and triacylglycerol levels increased to gestation day 19, then decreased to term. By the 6th h after birth, plasma and liver cholesterol and triacylglycerol levels had increased markedly. By 48 h after birth, the high hepatic cholesterol content was associated with an increase in the cholesteryl ester fraction. The microsomal cholesterol/phospholipid molar ratio decreased from gestation day 16 until 12 h after birth, then increased markedly from 36 to 48 h. There was an apparent inverse relationship between the change in microsomal cholesterol/phospholipid molar ratio and the fatty acid unsaturation index from gestation day 16 to 36 h after birth. The results suggest that in late gestation and before suckling, the low in vivo rate of hepatic cholesterol synthesis may not be due to low activity of HMG CoA reductase.     

Non-homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.     

Ontogeny of the GTP-Binding Protein Go in Rat Brain and Heart

We determined the ontogeny of the GTP-binding protein Go in rat brain and heart by employing highly sensitive enzyme immunoassay methods. In the brain, the alpha subunit of Go (Go alpha) gradually increased and reached adult levels approximately 20 and 30 days after birth in cerebral cortex and cerebellum, respectively. Concentrations of beta subunits, which were also quantified by the immunoassay, were almost equal to those of Go alpha in the brain of rats younger than 10 days, but were higher than those of Go alpha after 10 days. These results suggest that late development of GTP-binding proteins other than Go. Go alpha was immunohistochemically positive in neuropils and negative in cell bodies at any age tested. In the heart, the concentrations of Go alpha increased up to several times of the adult level just after birth, and then gradually decreased after the 20th postnatal day. The level of Go alpha in the liver, however, was very low and constant throughout ontogenic development. An immunohistochemical study indicated that Go alpha was positive in the cardiac muscle of young rat, but negative in that of adult rat. These results indicate that Go alpha exists in cells other than those of nervous tissues and neuroendocrine cells in some periods of ontogenic development.     

Postnatal Development and Possible Ligand Function of the CNS-Specific Membrane Glycoprotein CNSgp 130

CNSgp 130 is a CNS-specific membrane glycoprotein present in large amounts in the adult mammalian CNS. Using immunohistological techniques, we demonstrated that CNSgp130 is not detectable in the rat cerebellum at birth, and does not appear in the cerebellum until the tenth day of postnatal life. It is expressed first in the white matter of the cerebellar folia, and subsequently (by day 14) it is expressed also in the molecular layer. Expression in the granular layer is not seen until the 18th day of postnatal life, by which time the adult pattern of expression is established. CNSgp130 is also not detectable in the cerebrum at birth. However, it is expressed weakly but diffusely in the cerebrum by the fourth day of life. By the 10th day, there is strong expression in the cerebrum, in marked contrast to its virtual absence from the cerebellum at this stage. By quantitative absorption analysis, CNSgp 130 was undetectable on the day of birth, and increased steadily to 80% of adult values by the 22nd day of postnatal life. Binding studies with pure CNSgp130 demonstrated a Pronase-sensitive ligand in adult chicken brain. This ligand was absent from neonatal rat brain and non-CNS tissues.     

Developmental Changes in Rat Liver Xanthine Oxidase(s) and Its Intracellular Distribution

Developmental change and subcellular distribution of xanthine oxidase in the rat liver were examined.

The specific activity of the fetal liver xanthine oxidase increased sharply to the levels of the adult liver on the day of the birth. After birth, the activity dropped rapidly and on the 14th day after birth it was about 1/4 of adult level. Then the activity was regained and around 28th day after birth it was about the same as in adult level.

In the livers from 80 days old rats, about 60% of total xanthine oxidase activity was found in soluble fraction and the rest was distributed among particulate fractions including microsomal, lysosomal, mitochondrial and nuclear fractions.

In contrast to the adult livers 80% of total xanthine oxidase activity in fetal liver was found to be in particulate fractions.

From kinetic studies of xanthine oxidases in particulate and soluble fractions it was suggested that xanthine oxidase in soluble fraction and xanthine oxidase in particulate fraction might be different in their natures of protein molecule.     

Perinatal changes in amino acid metabolism of rat brain,especially alanine and glutamic acid

The changes in both the levels of some free amino acids and their metabolism in the rat brain during the first 24 hr of postnatal life were studied. The content of glutamic acid decreased for the first 2 hr; it remained at the lowest level for the next 4 hr, when it began to increase. The content of alanine decreased for the first 6 hr and approached the adult level. Oxygen consumption, glucose oxidation, and pyruvate formation in the cerebral slices of the 24-hr-old rats were as much as 150% of that of the 19-day-old fetus. The distribution profile of radioactivity incorporated into the cerebral amino acids from the subarachnoid-injected [U¹⁴C]glucose was also changed. In the 2- and 6-hr-old rats, 50% of the total radio-activity recovered in the free amino acids was in alanine. Its rate decreased to 30% in the 24-hr-old and was 2% in the adult, while the radioactivity incorporated into glutamic acid increased. Alanine aminotransferase activity started to increase at birth and had the highest level at 24 hr after birth. It then decreased and finally reached the same level as shown at birth. However, aspartate aminotransferase increased during the first 6 hr after birth and did not change until the end of the first day of life.     

Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity.

1. The pattern of NADP-linked malic enzyme activity estimated in the whole brain homogenate did not parallel that found in liver of developing rat. 2. Studies on intracellular distribution of malic enzyme in brain showed that the mitochondrial enzyme increased about three-fold between 10th and 40th day of life. Thereafter, a slow gradual increase to the adult level was observed. 3. The extramitochondrial malic enzyme from brain, like the liver enzyme, increased at the time of weaning, although to a lesser extent. At day 5 the brain malic enzyme was equally distributed between mitochondria and cytosol. 4. During the postnatal development, the contribution of the mitochondrial malic enzyme in the total activity was increasing, reaching the value approx. 80% at day 150 after birth. 5. The increase with age of the malic enzyme specific activity was observed in both synaptosomal and non-synaptosomal mitochondria, the changes in the last fraction being more pronounced. 6. The activity of citrate synthase developed markedly between 10-40 postnatal days, increasing about five-fold, while the specific activity of the enzyme did change neither in the synaptosomal nor in non-synaptosomal mitochondria at this period. 7. We conclude that the changes in malic enzyme activity in the developing rat brain are mainly due both to the increase of mitochondrial protein content and to the increase of specific activity of the mitochondrial malic enzyme.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.     

STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study

Neurons of the mouse were labeled with [³H]thymidine during their prenatal period of proliferation. The ³H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the ³H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [³H]β self-absorption in the material of the sections. Injection of a high dose of [³H]thymidine into young adult mice did not result in ³H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.