A neural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene.

In the present study we examined the methylation status of the glial fibrillary acidic protein (GFAP) gene promoter, analyzing various CG sites in both the human and rat gene in GFAP-expressing and nonexpressing tissues. Moreover, we studied the methylation of specific CG sites in different rat brain areas during postnatal development, in cell cultures highly enriched in specific neural- or non-neural-cell types (fibroblasts), and in human gliomas. The obtained results do not support a simple correlation between demethylation and expression of the GFAP gene but help to identify a cluster of CG sites in the 5'flanking region (from -1176 to -1471 in the rat) that are hypomethylated in neural cell types and localized in a region highly conserved between rat, mouse and human GFAP promoters. Neural-specific hypomethylation of this conserved zone can be observed also in the human GFAP gene both in normal brain tissue and neoplastic glial cells. A higher demethylation of the -1176 site at early stage of postnatal life was observed in specific rat brain areas, such as hippocampus and cerebellum. The most dramatic differences were observed in the cerebellum where a peak of demethylation of the -1176 site was detected at 15 days of postnatal life, followed by an intense remethylation of this site. Results of experiments in the CG4 glial progenitor cell line showed that demethylation of the -1176 site is already established before transcriptional activation of the GFAP gene. Moreover, results of experiments in primary cell cultures show that in neuronal cell types, such as cerebellar granule cells and embryonic cerebral hemisphere neurons, the level of demethylation of the -1176 site is comparable to that observed in cultured astrocytes. In contrast a high level of methylation can be observed in cultured non-neural cell types (fibroblasts). Such neural-specific hypomethylation could be established in a very early stage in the progression along the neural cell lineage and could play a role in maintaining a local open chromatin conformation which is then necessary to allow the interaction with specific regulatory factors present in astroglial cells.

Obligatory role of IFN-gamma in induction of lymphokine-activated and T lymphocyte killer activity, but not in boosting of natural cytotoxicity.

The biological role of the murine IFN-gamma endogenously secreted during cell activation has been probed by the use of a rat mAb (AN18) that specifically neutralizes its activity. When An18 mAb is added to the cultures of BALB/c and C57BL/6 nylon nonadherent spleen cells (naSpc) stimulated for 18 h with 1000 U of IL-2, the normally released IFN-gamma can no longer be detected in the supernatants and the IL-2-induced proliferative response is markedly reduced as compared with control cultures set up in the presence of an unrelated rat mAb. By contrast, the increased natural cytotoxicity is not affected. The 96-h culture of both BALB/c and C57BL/6 naSpc in the presence of 1000 U of IL-2 resulted in marked lymphocyte proliferation and generation of lymphokine-activated killer activity. The presence of An18 mAb strongly inhibited both functions. Similarly, when naSpc were stimulated with mitomycin C-inactivated allogeneic leukocytes in the presence of An18 mAb, the normal proliferative response and specific cytotoxicity were almost abolished, whereas the secretion of IL-2 was in no way affected. Lymphocytes recovered from 3-day cultures stimulated by IL-2 or allogeneic cells in the presence of An18 mAb displayed a decrease of the expression of the p55 chain of IL-2R, as shown by flow cytofluorimetry. Moreover, binding experiments with 125I-labeled IL-2 showed that lymphocytes from allostimulated cultures set up in the presence of An18 mAb display a decreased number of low affinity and almost no high affinity IL-2R as compared with control cultures. These data show that endogenous IFN-gamma plays an obligatory role for the de novo induction of cytolytic activity in lymphokine-activated killer cells and CTL, most probably by affecting the membrane expression of high affinity IL-2R.

Changes of synaptosomal energy metabolism induced by hypoxia during aging.

Synaptosomes were isolated from the motor area of the cerebral cortex of normoxic or hypoxic (PaO2 = 17-19 mmHg, for 15 min) beagle dogs of different ages. Synaptosomes were incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24 degrees C) and the energetic state was defined by: the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate); the respiratory rate; the redox state of the intramitochondrial NAD-couple. By the present experimental model, it is possible to evaluate the potential damage (induced by the "in vivo" hypoxic insult) that synaptosomes cannot reverse under optimal incubation. Aging affected the phosphorylation state of the post-hypoxic incubated synaptosomes. The oxygen consumption rate was quite similar in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, but the cytochrome c and a contents were lower in the preparations from hypoxic older brains. In dogs of different ages, hypoxia always lowered the respiration of the synaptosomes, but aging affected the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. The synaptosomal energetic state was defined also by the redox state of the intramitochondrial NAD-couple (delta Gox-red) and the phosphorylation state of adenine nucleotide system (delta GATP). The free-energy change (delta delta G) for the coupled reactions was calculated, too. In synaptosomes isolated from the cerebral cortex of dogs submitted to hypoxia, the equilibrium (calculated for the mitochondrial electron transfer chain and the phosphorylation of adenine nucleotides) was markedly altered as function of aging.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures.

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-3H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.

Acetylation and phosphorylation of histones and nonhistone chromosomal proteins in neuronal and glial nuclei purified from cerebral hemispheres of developing rat brain.

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl-CoA and of [gamma-32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low-molecular-weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000-90,000 daltons)--absent at 1 day, appearing at 10 days, and more evident at 30 days of age--was observed in both neuronal and glial nuclei.

Remodeling and sorting process of ethanolamine and choline glycerophospholipids during their axonal transport in the rabbit optic pathway.

The existence of a mechanism by which the ester- and ether-linked aliphatic chains of the major phospholipids are retailored during their axonal transport and sorted to specific membrane systems along the optic nerve and tract was investigated. A mixture of [1-14C]hexadecanol and [3H]arachidonic acid was injected into the vitreous body of albino rabbits. At 24 h and 8 days later, the distribution (as measured by the 3H/14C ratio) and the positioning (as monitored by hydrolytic procedures) of radioactivity in the various phospholipid classes of retina, purified axons, and myelin of the optic nerve and tract were determined. At the two intervals after labeling, the 3H/14C ratios of each diradyl type of phosphatidylethanolamine and phosphatidylcholine were (a) substantially unchanged all along the axons within the optic nerve and tract and (b) markedly modified in comparison with those found in the retina and axons for molecular species selectively restricted to myelin sheath. Evidence is thus available that intraxonally moving ethanolamine and choline glycerophospholipids, among others, are added to axonal membranes most likely without extensive modifications. In contrast, they are transferred into myelin after retailoring. Through these two processes, the sorting and targeting of newly synthesized phospholipids to their correct membrane domains, such as axoplasmic organelles, axolemma, or periaxonal myelin, could be controlled.

Methylation of chromosomal proteins in neuronal and glial nuclei purified from cerebral hemispheres of rat during postnatal development.

The process of methylation of chromosomal proteins [histones and nonhistone proteins (NHP)] in neuronal and glial cell nuclei obtained from cerebral hemispheres of rats at 1, 10, and 30 days of age was investigated. Purified neuronal and glial nuclei were incubated in the presence of S-adenosyl[methyl-3H]methionine. Histone and NHPs were extracted and fractionated by polyacrylamide gel electrophoresis. The results obtained indicate remarkable differences in the process of methylation of histones and NHPs between neuronal and glial nuclei, especially during the first period of postnatal development. In both nuclear populations the histone fraction H3 was labeled to a greater degree than the other fractions and showed the major changes during postnatal development. The densitometric and radioactive patterns of NHPs show considerable changes in the two nuclear populations at the various ages examined. The main difference between neuronal and glial nuclei consists in the intense methylation of proteins with a molecular weight of approximately 100,000, which are present in neuronal nuclei and virtually absent in glial ones. The results obtained may be correlated with the different chromatin structures of neuronal and glial nuclei and with the patterns of maturation and differentiation of neuronal and glial cells during postnatal development.

Influence of aging and exogenous substances on cerebral energy metabolism in posthypoglycemic recovery.

In rats of different ages, acute severe hypoglycemia with isoelectric EEG induced extensive deterioration of the energy state and gross alteration of amino acid contents. During recovery of adult animals, tissue glucose concentration returned to normal, while the rate of glycogen synthesis was slow, both lactate and pyruvate concentrations increasing above normal. In the recovery period of "adult" rats, the ATP concentration increased but the adenine nucleotide pool remained reduced, even if the ADP and AMP concentrations were close to normal. Phosphocreatine was restored to normal concentrations with reciprocal changes in creatine content. In adult rats, during the recovery there was a rise in glutamate and glutamine concentrations, gamma-aminobutyrate concentration returning to normal value. Ammonia and aspartate decreased below normal, while alanine increased above normal. Aging does not affect the cerebral metabolic derangement occurring in severe hypoglycemia, but rather the metabolic changes that the brain tends to reverse during the posthypoglycemic restitution. In fact, there was lower restitution of the concentrations of cerebral cortical metabolites of "mature" and "senescent" rats in comparison with "adult" ones. Particularly, in older brains the concentrations of many amino acids and adenylate nucleotides remained largely abnormal. The effect of some agents on the posthypoglycemic recovery was tested: (a) dihydroergocristine; (b) eburnamonine; (c) raubasine; (d) almitrine; (e) piracetam. During the posthypoglycemic recovery, these different agents exhibited different interferences on glycolytic metabolites, amino acids and energy-rich phosphates. However, a more limited effect of the tested agents, which decrease with aging, was observed.

Effect of hypothyroidism on the biogenesis of free mitochondria in the cerebral hemispheres and in cerebellum of rat during postnatal development.

The effect of propylthiouracil-induced neonatal hypothyroidism on some aspects of the biogenesis of free (non-synaptosomal) mitochondria in the cerebral hemispheres and in the cerebellum of developing rat has been studied. The results obtained show that in hypothyroid rats mitochondrial DNA synthesis is delayed, mitochondrial RNA synthesis is not affected and cytochrome aa3 content of mitochondria is lower than in controls. Furthermore ultrathin sections of 14- and 21-day old hypothyroid rat cerebella show mitochondria with an altered ultrastructural organization and large intracristal spaces.

Synthesis of myelin proteins and ultrastructural investigations in regenerating rat sciatic nerve.

Myelin protein synthesis, as well as ultrastructural and morphometric changes in regenerating peripheral nerve, was studied. Sciatic nerves of rats were crushed unilaterally; sham-operated nerves of the contralateral side served as controls. For the in vivo experiments, rats were killed at selected periods after the nerves were crushed (30, 60, 90, and 120 days); seven days prior to killing, the animals were injected intravenously with L-[4,5-3H]leucine. For the in vitro experiments, proximal and distal segments of sciatic nerve and equivalent sham-operated nerves were labeled with 3H-amino acid mixture 90 days after axotomy. Purified myelin was isolated from nerve segments; specific radioactivity and gel electrophoretic patterns of proteins were analyzed. Cross-sectional electron microscope (EM) preparations of proximal, distal, and contralateral segments of nerves also were examined. Results showed that the incorporation of labeled amino acids into total myelin proteins was enhanced significantly in the distal segment of sciatic nerves at all of the periods of regeneration studied. The yield of myelin protein per mm distal nerve segment increased as regeneration proceeded. The remyelination of fibers early after nerve crush was weak, whereas it gradually attained the normal range 90-120 days after axotomy. Morphometric analysis of myelin sheath thickness of regenerating axons was consistent with the data obtained for myelin protein synthesis.

Effect of hypothyroidism on the labeling of the various RNA species in developing rat brain.

The effect of hypothyroidism on the vitro incorporation of [3H]uridine into different RNA species in tissue slices of rat cerebral hemispheres at 5, 10, 15, and 21 days of age has been investigated. Gel electrophoresis analysis of total, nuclear and microsomal RNA was also accomplished. The results obtained indicate that RNA labeling is differently influenced by hypothyroidism at the various ages examined. RNA labeling is not significantly affected at 5 days of age while at later ages and especially at 21 days it is higher in hypothyroid rats compared to the controls. Moreover distinct differences at the various ages in the transport of newly synthesized RNA from the nucleus to the cytoplasm in the two groups of animals were found. These results are in agreement with the hypothesis that thyroid hormone deficiency causes a delay of the processes of cell proliferation and differentiation in developing rat brain.

Determination of C20-C30 fatty acids by reversed-phase chromatographic techniques: an efficient method to quantitate minor fatty acids in serum of patients with adrenoleukodystrophy.

An analytical method for the determination of saturated very long chain (VLC) fatty acids in the serum has been devised. Free fatty acids obtained after hydrolysis of total lipid extracts were converted into p-bromophenacyl esters. The derivatives were purified in two sequential steps by clean-up on C18 reversed-phase cartridge and fractionation by reversed-phase thin-layer chromatography (TLC), and then quantitated by high performance liquid chromatography (HPLC) analysis. This technique provides a reliable and alternative method for the biochemical identification of patients and carriers of an inherited metabolic disease characterized by the accumulation of saturated VLC fatty acids (C24-C26) such as Adrenoleukodystrophy (ALD). In four cases of diagnosed ALD the fatty acid composition of serum total lipids was dramatically enriched in saturated VLC fatty acids compared to controls. The ratio of hexacosanoic acid (C26:0) to docosanoic acid (C22:0) in ALD patients was approximately six-fold higher than that of healthy controls or patients affected by metabolic or neurological disorders other than ALD.

RNA synthesis in neuronal and glial cell nuclei from rat cerebral hemispheres during early postnatal development.

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [3H]uridine) and in vitro (either by incubation of tissue slices with [3H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg2+-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn2+-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.

Myelination process in the rat sciatic nerve during regeneration and development: molecular species composition and acyl group biosynthesis of choline-, ethanolamine-, and serine-glycerophospholipids of myelin fractions.

The content of alkenyl-acyl, alkyl-acyl and diacyl types of the three major myelin glycerophospholipids such as PtdCho, PtdEtn and PtdSer was determined in myelin fractions prepared from sciatic nerve segments of rats at 12, 25 and 45 days after birth, and of adult rats (6-month-old) 90 days after crush injury. The biosynthesis and metabolic heterogeneity of lipid classes and types were also studied by incubation with [1-14C] acetate of nerve segments of young rats at different ages as well as crushed and sham-operated control nerve segments of adult rats. The analysis of composition and positional distribution in major individual molecular species extracted from light myelin and myelin-related fraction suggest that the metabolism of alkenyl-acyl-glycerophosphorylethanolamines and unsaturated species of PtdCho and PtdSer may not be regulated in the same manner during peripheral nerve myelination of developing rat and remyelination of regenerating nerve in the adult animal. The 14C-radioactivity incorporation into lipid classes and alkyl and acyl moieties of the three major phospholipids of sciatic nerve segments during the developmental period investigated revealed that Schwann cells were capable of synthesizing acyl-linked fatty acids in both myelin fractions at a decreasing rate and with different patterns during development. In regenerating sciatic nerve of adult animals the labeling of myelin lipid classes and types of remyelinating nerve segment distal to the crush site was markedly higher than that of sham-operated normal one; however, the magnitude and the pattern of the specific radioactivity never approached those observed during active myelination of the nerve in young animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aging on cerebral cortex energy metabolism in hypoglycemia and posthypoglycemic recovery.

Severe hypoglycemia, causing the cessation of spontaneous EEG, induced in cerebral cortex of rats of different ages, causes gross energy failure and extensive derangement of both carbohydrate and amino acid contents. During posthypoglycemic recovery of adult rats, there was moderate restitution of energy metabolism and both ATP concentration and adenine nucleotide pool remained still reduced, even if the creatine phosphate and ADP contents were close to normal. During recovery of adult rats there was a rise in glutamate and glutamine concentrations and the perturbated aspartate and gamma-aminobutyrate cerebral contents normalized. Ammonia content decreased to normal, while alanine content was markedly elevated. Aging does not affect the cerebral metabolic derangements occurring in severe hypoglycemia, but rather the metabolic changes that the brain tend to reverse during the posthypoglycemic restitution. In fact, there was lower restitution of the contents of cerebral cortical metabolites of "mature" and "senescent" rats in comparison with "adult" ones. Particularly, in older brains the contents of many amino acids and adenylate nucleotides remained largely abnormal.

Influence of aging on cerebral derangement by acute severe hypoxia during hypovolemic hypotension.

In synaptosomes isolated from the motor area of the cerebral cortex of beagle dogs and incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24 degrees C), the energetic state was defined by the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate), the redox state of the intramitochondrial NAD-couple, and the respiratory rate. By the experimental model utilized in the present research, it is possible to evaluate the potential synaptosomal damage induced by the in vivo hypoxic insult. Aging affects the phosphorylation state of the posthypoxic incubated synaptosomes. Although the oxygen consumption rate is the same in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, the cytochrome c and a contents are lower in the preparations from older brains. This points to higher activity of cytochromes in the synaptosomes from "mature" and "senescent" hypoxic animals. In dogs of different ages, hypoxia lowers the respiration of the synaptosomes but aging affects the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. In synaptosomes isolated from older hypoxic brains, the free energy utilized for the synthesis of two moles of ATP (delta GATP) is progressively lower than that released upon the transfer of electrons from the NADH to cytochrome c (delta Gox-red).

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development.

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.

Labeling of RNA in young and adult rat brain: evidence for different RNA processing.

The labeling of RNA in young and adult rat brain has been studied by measuring in vitro (tissue slices incubation) the incorporation of labeled uridine into RNA of total tissue and of the various subcellular fractions purified from cerebral hemispheres of 1- and 10-month-old rats. Gel electrophoretic analysis of the newly synthesized nuclear and microsomal RNA was also accomplished. An active metabolism of RNA in adult animals was found; moreover, distinct differences in ribosomal RNA processing in cerebral hemispheres of 1- and 10-month-old rats, with a more rapid processing in the brain of adult animals, were obtained.

Posttranslational modifications of nuclear proteins in rat cerebral hemispheres during postnatal development.

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.