5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.     

Evolution of 3-hydroxy-3-methylglutaryl-CoA reductase and microsomal membrane fluidity throughout chick embryo development

The pattern of chick liver and brain 3-hydroxy-3-methylglutaryl-CoA reductase and its relationship with changes in microsomal membrane fluidity was studied during embryonic and postnatal development. A peak of brain activity was found at 19 days of embryonic development, while liver activity only increased after hatching. A significant increase in cholesterol content of brain microsomes occurred at about 14 days of incubation, decreasing afterwards. No significant variations were observed in liver microsomes during the same period. A similar profile was found in the phospholipid content of both brain and liver microsomes. The cholesterol/lipidic phosphorus molar ratio of brain and liver microsomes did not exhibit significant changes throughout embryonic and postnatal development. These results demonstrate that membrane-mediated control does not regulate the evolution of reductase activity during this developmental period.     

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging

Brain development and aging are associated with alterations in multiple epigenetic systems,including DNA methylation and demethylation patterns.Here,we observed that the levels of the 5-hydroxymethylcytosine (5hmC) ten-eleven translocation (TET) enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews (Tupaia belangeri chinensis).The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development,whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews,including in the prefrontal cortex and hippocampus,but not the cerebellum.Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels.Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.     

A comparison by species, age and sex of cysteinesulfinate decarboxylase activity and taurine concentration in liver and brain of animals

Cysteinesulfinate decarboxylase activity and taurine concentration were determined in liver and brain of rats, mice, cats, guinea-pigs and sheep. Values were compared for male and female animals and in some cases measurements were also made in animals of different ages. Cysteinesulfinate decarboxylase activity and taurine concentration were also measured in liver and brain of male and female rat pups during the postnatal period. Hepatic cysteinesulfinate decarboxylase activity increased in both male and female rat pups during the postnatal period and then declined markedly in female rats so that activity in adult males was 16-fold that in adult females. Cysteinesulfinate decarboxylase activity in liver of 5- to 6-week old kittens was 73 times that observed in liver of 15-month old cats. Taurine level in liver of guinea-pigs was much lower than that in liver of any other species studied.     

DNA synthesis in the brain and liver of rats in various stages of postnatal development]

The intensity of incorporation of 3H- and 14C-thymidine in the brain and liver DNA in rats of different ages was investigated. It was proved that both the replicative and oxyurea-resistant DNA synthesis might proceed in the rat brain cells. The intensity of these processes changes sharply during postnatal development.     

Methamphetamine-enhanced embryonic oxidative DNA damage and neurodevelopmental deficits

Methamphetamine (METH) causes dopaminergic nerve terminal degeneration and functional deficits in adult mice, but its neurodevelopmental effects are unclear. We investigated METH-initiated oxidative DNA damage in brain during the embryonic and fetal periods, and the postnatal histological and functional consequences. Pregnant CD-1 mice were treated with a single dose of METH (20 or 40 mg/kg ip) or its saline vehicle on Gestational Day 14 or 17. METH enhanced conceptal DNA oxidation, determined by 8-oxoguanine formation, in brain and liver by at least 2-fold at 1 h (P < 0.05), and more so in some fetal brains at 4 h. After birth, motor coordination on the rotarod apparatus in the METH-exposed offspring was impaired for at least 12 weeks (P < 0.05). Unlike in adults, this postnatal functional deficit in offspring exposed in utero to METH was not associated with degeneration of striatal dopaminergic nerve terminals at 12 weeks of age determined by tyrosine hydroxylase staining, suggesting a novel pathological mechanism in utero. This is the first evidence of oxidative DNA damage in embryonic and fetal brain caused by amphetamines, leading to long-term postnatal neurodevelopmental deficits via a mechanism different from that underlying the neurodegeneration observed in METH-exposed adults.     

Synthesis of chain elongated-desaturated fatty acids from palmitic acid by liver and brain microsomes during the development of the pig

Rates of chain elongation-desaturation of C16:0 were compared for liver and brain microsomes in fetal, neonatal and postnatal piglets. Rates of chain elongation of C16:0 were greatest in liver in the postnatal period. For all developmental periods examined, the amount of chain elongated fatty acid of C20 or greater chain length exceeded the amount of C18 precursors synthesized from C16:0. These observations suggest that chain elongation of fatty acids occurs during the latter part of gestation at rates equivalent to that observed in the early postnatal period.     

THE EFFECT OF AGE, GROWTH RETARDATION AND ASPHYXIA ON ASCORBIC ACID CONCENTRATIONS IN DEVELOPING BRAIN

Abstract— The concentration of ascorbic acid in whole rat brain during the first week of postnatal life was up to 100 per cent higher than in adult animals. A progressive fall in concentration occurred between 4 and 30 days of age. Corresponding changes did not occur in liver and adrenal gland, two other organs rich in ascorbic acid. Rats subjected to growth retardation during the fetal and suckling periods had, at 25 days of age, levels of ascorbic acid in the cerebellum and brainstem significantly higher than those of control animals. A period of prolonged asphyxia in 5-day-old rats resulted in a significant 4 per cent reduction in whole brain ascorbic acid concentration.     

High-performance liquid chromatographic analysis of methylation changes of CCGG sequence in brain and liver DNA of mice during pre- and postnatal development

The change of the methylation of CpG in the CCGG sequence of brain and liver DNAs of mice during late fetal and suckling periods was determined by high-performance liquid chromatography using a reversed-phase column and 0.1 M phosphate buffer (pH 6.0) as the mobile phase. The tissue DNA was digested with the restriction enzyme, MspI, and was labeled at the 5′-end with [γ-³²P]ATP. The cpm% of deoxycytidine 5′-monophosphate (5mdCMP) in total CpG dinucleotides was calculated from the equation 5mdCMP/total CCGG (cpm%) = (5mdCMP)_MspI,cpm/{(5mdCMP)_MspI,cpm + (dCMP)_MspI,cpm} × 100. The brain DNA exhibited a significant decrease in CpG methylation at prenatal day 18 but little change after birth. This marked decline of 5mdCMP in the CCGG sequence may be associated with the increase of enzymes before birth. The liver DNA showed considerable change during the late prenatal period. The observed changes of CpG methylation in liver DNA are indicative of the corresponding alterations of enzymes, multinucleate cells and hepatocytes. The results obtained indicate that both brain and liver cells have the development-associated changes in the conformation and transition of DNA around the time of birth.     

Ontogenesis of the insulin receptor in the rabbit brain

We delineated the ontogeny of the brain insulin binding, insulin receptor number and affinity using plasma membranes isolated from the rabbit. Specific 125I-insulin binding and receptor number expressed per milligram of protein increased from the 20 day gestation fetus to the 1-day-old newborn, declining thereafter to attain adult values by day 6 of postnatal life. Specific 125I-insulin binding and the receptor number in the adult brain was less than the fetal and neonatal (1 day) brain receptors. Although a similar trend was observed specifically during fetal development, the changes in receptor number expressed per microgram DNA were not significant in the neonatal period. The adult brain insulin receptor number was higher than the 20- to 27-day fetus and similar to that of the 30-day fetus and the 1- to 5-day newborns. The total receptor number correlated linearly with the brain plasma membrane protein increment velocity. The affinity of the receptors increased during early fetal development (20-27 days) and remained constant thereafter in the postnatal period. We conclude that the ontogenic changes of the brain insulin receptors are similar to the ontogenic changes of brain plasma membrane protein. The developmental changes are more pronounced when the receptor number is expressed per milligram protein versus microgram DNA.     

Juvenile toxicity assessment of open-acid lovastatin in rats

BACKGROUND: Lovastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase, reduces de novo cholesterol biosynthesis primarily in the liver. Since cholesterol is a major component of brain myelin and peak periods of brain myelination occurs after birth, this study was designed to encompass this period in rats and evaluate the potential neurotoxic effects. METHODS: The pharmacologically active, open‐acid form of lovastatin was administered to groups of 50 Sprague–Dawley rats per sex subcutaneously once daily at dose levels of 0 (vehicle), 2.5, 5, or 10 mg/kg/day beginning on postnatal day 4 and continuing until termination on postnatal day 41 to 51. Physical signs and body weights were monitored during the study. Animals were assessed in a battery of behavioral tests, and at termination a set of animals were examined for gross and histological changes. RESULTS: There were no test article‐related deaths, physical signs, or effects on preweaning and postweaning body weights during the study. In the behavior tests there were no test article‐related effects in the passive avoidance, auditory startle habituation, open‐field motor activity, or FOB. No test article‐related postmortem findings were observed, including brain weights and histomorphology of brain, spinal cord, eye, optic nerve, or peripheral nerve. CONCLUSION: Based on these results, the no‐effect level for general and neurobehavioral toxicity in neonatal rats was ≥10 mg/kg/day for open‐acid lovastatin. Birth Defects Res (Part B) 92:314–322, 2011. © 2011 Wiley‐Liss, Inc.     

Changes in chick liver and brain mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase during development

Phosphorylation and decarboxylation of mevalonate in chick liver and brain was investigated during early post hatching stages of development. In chick liver, both mevalonate kinase and mevalonate-5-phosphate kinase increased their activity from day 5 of age while pyrophosphate decarboxylase activity remained low during the first days after hatching, increased sharply up to day 9 of age, and remained practically unchanged thereafter. The developmental pattern obtained in brain shows a slight decrease in the phosphorylation and decarboxylation of mevalonate after the first week of postnatal development. Further studies were performed using the specific substrate of mevalonate-5-pyrophosphate decarboxylase, corroborating the results obtained using mevalonate as substrate. Changes in hepatic decarboxylase were more pronounced than those observed in mevalonate-phosphorylating enzymes, thus suggesting an important role for decarboxylase in the control of cholesterogenesis during postnatal development.     

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.     

Cerebral myelinogenesis in theSnell dwarf mouse: Stimulatory effects of GH and T4 restricted to the first 20 days of postnatal life

We attempted to define the critical time period during early postnatal life when GH and T₄ are essential for myelination. We administered bGH and T₄ toSnell dwarf mice during the first and second 20 days after birth. Positive results were obtained only when hormones were given during the first 20 days of postnatal life. We observed a distinct increase in brain weight, DNA content, CNPase activity and a remarkably increased level of spontaneous locomotion activity with a diurnal periodicity. Morphological observations of brain sections stained for myelin basic protein (MBP) correlated the biochemical findings. The later administration of hormones was ineffective. Our interpretation is that the administration of exogenous hormones led to increased myelinogenesis through their stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.     

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits during embryonic and early postnatal development of rat spleen and liver was studied in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The distribution of LMP7 and LMP2 immune subunits in spleen and liver cells was also evaluated throughout development. The common tendency of both organs to increase the expression of both LMP7 and LMP2 subunits on the 21st postnatal day (P21) was found. However, the total proteasome level was shown to be constant. At certain developmental stages, the dynamics of immune subunits expression in the spleen and liver was different. While the gradual enhancement of both immune subunits was observed on P1, P18 and P21 in the spleen, the periods of gradual increase observed on E16 (the 16th embryonic day) and E18 gave way to a period of decrease in immune subunits on P5 in the liver. This level did not reliably change until P18 and increased on P21. The revealed changes were accompanied by an increase in chymotrypsin-like activity and a decrease in caspase-like activity in the spleen at P21 compared to the embryonic period. This indicates the increase in proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased compared to the embryonic period by P21. The dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Moreover, it was discovered that the increase in the expression of immune subunits during early spleen development is associated with the process of formation of white pulp by B- and T-lymphocytes enriched with immune subunits. In the liver, the increase in the level of immune subunits by P21 was also accompanied by an increase of their expression in hepatocytes. While the decrease of their level by P5 may be associated with the fact that the liver has lost its function as the primary lymphoid organ in the immune system by this time, as well as with the disappearance of B-lymphocytes enriched with immune proteasomes. In the spleen and the liver, MHC class I molecules were found during the periods of increased levels of proteasome immune subunits. On E21, the liver was enriched with neuronal nitric oxide synthase (nNOS); the level of nNOS decreased after birth and then increased by P18. This fact indicates the possibility of the induction of expression of the LMP7 and LMP2 immune subunits in hepatocytes via a signaling pathway involving nNOS. These results indicate that compared to the rat liver cells, splenic T cell immune response develops in rats starting around P19–P21. First, a T-area of white pulp is formed in the spleen during this period. Second, an increased level of immune proteasomes and MHC class I molecules in hepatocytes can ensure the formation of antigenic epitopes from foreign proteins and their delivery to the cell surface for subsequent presentation to cytotoxic T-lymphocytes.