Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue

A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

Soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities in subcellular fractions of young and adult rat brains

The subcellular distribution of soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities were analyzed in one and five month old rat brains using Leu- and Arg-2-naphthylamide as substrates. Both soluble leucyl- and arginyl-aminopeptidase activities showed the highest levels in the synaptosomal fraction in the two groups of rats. The highest levels of membrane-bound leucyl- and arginyl-aminopeptidase activities were found in the microsomal fraction in the two ages studied. There were no differences between the two ages in soluble leucyl- and arginyl-aminopeptidase activities. However, a significant decrease in both membrane-bound enzymatic activities was evidenced in the synaptosomal fraction of older rats. Developmental changes of these aminopeptidase activities in a determined subcellular localization, may reflect modifications in their effect on neuropeptides susceptible to be hydrolyzed in this particular location.     

Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient

A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 μm pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between δ1.050 and δ1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia.A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera.The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.     

A particulate DNA polymerase activity in adult rat brain

A particulate fraction of adult rat brain (sucrose buoyant density 1.24 gm/ml) catalyzed the incorporation of [³H]dTTP into an acid-insoluble product in an endogenously templated reaction sensitive to ribonuclease pretreatment. Upon fractionation, this activity was identified in the cerebellum, pons, frontal lobes and base. The DNA polymerase present in these brain fractions exhibited a strong preference for the synthetic template dT_12–18·poly rA rather than dT_12–18·poly dA; dT₁₀ was completely inactive. Purification and equilibrium Cs₂SO₄ gradient centrifugation of the [³H]DNA product-endogenous template complex suggested that RNA was serving as primer for endogenous DNA synthesis.     

RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5-triphosphates and Mg²⁺. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs₂SO₄ equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60 C. This DNA product did not hybridize with poly(A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.This work was supported in part by a contract with the U.S. Department of Energy and a grant from the U.S. Naval Research.     

Changes in some enzymic activities of separated neuronal and glial cell-enriched fractions from rat brains during development

Bulk-prepared neuronal perikarya and glial fractions have been used to study developmental changes of some enzymic activities concerning glycolysis and synthesis of neurotransmitters. Somewhat higher pyruvate kinase activity was found in neuronal perikarya than in glial cells, and its rapid rise was observed during early developmental stages. Increased K⁺ concentration ord-glutamate addition to the incubation medium enhanced consumption of phosphoenolpyruvate. This activation of the enzyme was small just after birth, but it increased in parallel with development to adult level, where the activation in glial fractions was over twice that in neuronal fractions. Choline acetyltransferase activity was found in purified neuronal fractions and increased with age; glutamate decarboxylase was also found in high activity in purified neuronal fractions and increased with development. However, some enzymic activities were also found in glial fractions, and possibilities of contamination by synaptosomal, myelin, or other subfractions are discussed.     

Colonic cell proliferation and growth fraction in young,adult and old rats

Colonic epithelial proliferation was investigated in three groups of rats, aged 3, 60 and 121 weeks. As reported in previous work, the crypts were markedly longer in the young rats, and the number of labelled cells per crypt was significantly greater. There was an upward movement of the marker positions derived from the distribution of labelled cells within the crypt of the young rats. This was a consequence of the increased crypt length, so that the growth fraction, as expressed as a percentage of crypt length, was the same. The proliferative changes between the young rats and the other aged rats were therefore effected by altering the size of the crypts, while maintaining the kinetic organisation. There was no evidence of any proliferative changes or changes in the growth fraction when the colons of the old rats were compared with those of the 60 week old rats.     

RNA polymerase I and II activities in different brain regions of young, adult, and old rats

The activities of RNA polymerase I and II were assayed in nuclei isolated from different regions (cerebral cortex, cerebellum, hypothalamus, hippocampus, corpus striatum and pituitary) of brains from young (10 days), adult (6 months), and old (2 years) rats. The RNA polymerases I and II activities generally increased during maturation, i.e., from 10 days to 6 months of postnatal age and then showed a decrease from 6 months to 2 years of age in all the regions except in cerebral cortex where the RNA polymerase II activity was highest at 10 days but showed a gradual decrease through the lifespan up to 2 years.     

The effects of heparin on endogenous DNA polymerase activity of rat liver nuclei and chromatin fractions

1. 1. Heparin activates endogenous DNA polymerase activity in isolated rat hepatic nuclei to a synthesis rate two to three times the control values, when added in amounts as small as 10% of total nuclear DNA.

2. 2. Cations such as polylysine, polyornithine and unfractionated histones form insoluble complexes with heparin and reverse its effect in either order of addition.

3. 3. Autoradiography demonstrates many apparent new sites of DNA synthesis in heparin-treated nuclei, both in previously inactive and in previously active nuclei.

4. 4. Electron microscopy shows a dramatic change in the chromatin pattern upon treatment with heparin; fiber diameters are significantly decreased, probably indicating loss of supercoiling.

5. 5. The response to heparin of the endogenous polymerase activity in three chromatin fractions is examined. Two of the three fractions show patterns similar to whole nuclei; the third, a high specific activity fraction, has a distinctively different pattern of response to heparin.

6. 6. The kinetic data combined with the behavior of the high specific activity chromatin fraction indicate that the heparin effect on endogenous DNA synthesis may have more than one component.

     

CNPase activity in myelin from adult rat brains after prolonged lead exposure in vivo

The present study investigated the sensitivity of rats cerebral myelin to prolonged toxicity of lead (Pb) that imitates environmental exposure to this metal. The results indicated that 90 days exposure of young adult rats to lead in drinking water affects the morphology of myelin sheaths, expressed in disintegration of its multilamellar structure. Both, the protein content and the activity of the myelin-specific enzyme CNPase (2',3'-cyclic nucleotide 3-phosphodiesterase), were lowered. The Michaelis-Menten kinetic for CNPase in myelin obtained from control and Pb-treated rats was different. Km increased and Vmax decreased when compared to controls. Observed disturbances in enzyme activity may be one of the potential reasons of the ultrastructural changes. It is thus tempting to speculate that Pb may be considered as a one of the factors contributing to demyelinating diseases.     

Lysosomal hydrolases in neuronal, astroglial, and olidodendroglial enriched fractions of rabbit and beef brain.

Arylsulfatases A, B, and C, beta-galactosidase, and acid phosphatase were assayed in neuronal, astroglial, and oligodendroglial fractions isolated from adult rabbit and beef brains. The specific activities of all acid hydrolases were lower in beef cells compared to rabbit cells. The lysosomal enzymes of the rabbit neuronal fraction showed 10--25 time higher activities than the oligodendroglial fraction and 5-fold higher activities than the astroglial fraction. In beef brain, the specific activities of these enzymes were similar in oligodendroglia and astrocytes but 4--10 times lower than in neurons. The low activity of arylsulfatase A and beta-galactosidase in oligodendroglial cells may suggest that the low turnover of cerebroside and sulfatide in myelin may be regulated in part by the enzymes that catalyze their degradation.     

Characteristics of the DNA polymerase beta-directed unscheduled DNA synthesis in isolated nuclei from adult rat liver

Isolated nuclei from adult rat liver have been used as a model system to define several characteristics of the unscheduled DNA synthesis supported by DNA polymerase beta. Many of these characteristics have been found to reflect some catalytic properties (pH optimum, divalent cations requirement, dependence on all four deoxyribonucleoside triphosphates, apparent Km for dTTP) as well as sensitivity to various agents (differential inhibitors of eukaryotic DNA polymerases, phosphate, DNA intercalating drugs, chemical or thermal denaturation) commonly regarded as typical of DNA polymerase beta itself. Given the new picture of the enzymology of DNA repair synthesis which has recently emerged, none of the above characteristics seem to be suitable candidates as diagnostic tools of a repair polymerization process.     

Colonic cell proliferation and growth fraction in young, adult and old rats.

Colonic epithelial proliferation was investigated in three groups of rats, aged 3, 60 and 121 weeks. As reported in previous work, the crypts were markedly longer in the young rats, and the number of labelled cells per crypt was significantly greater. There was an upward movement of the marker positions derived from the distribution of labelled cells within the crypt of the young rats. This was a consequence of the increased crypt length, so that the growth fraction, as expressed as a percentage of crypt length, was the same. The proliferative changes between the young rats and the other aged rats were therefore effected by altering the size of the crypts, while maintaining the kinetic organisation. There was no evidence of any proliferative changes or changes in the growth fraction when the colons of the old rats were compared with those of the 60 week old rats.