Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei

Austoker, J.; Cox, David J.; Mathias, A. P.

doi:10.1042/bj1291139

Cited by 104 publications

(48 citation statements)

References 46 publications

(35 reference statements)

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“…Based on DNA determinations, this procedure yields about 75% of the nuclei found in a homogenate of DRG. The protein/DNA ratio was approximately 5.3:1 (23). Chromatin was prepared from purified nuclei according to Spelsberg and Hnilica (24), microsomes were isolated as described by Banerjee et al (14), and DNA was determined with the diphenylamine method described by Burton (25).…”

Section: Methodsmentioning

confidence: 99%

“…Cell nuclei, prepared by using ionic conditions and pH different from those of simple Triton extraction, were not distinguishable from crude Triton-extracted DRG with respect to saturability and affinity, confirming this conclusion. Although the purity of the nuclei, isolated as described in Materials and Methods, has been confirmed by biochemical and electron microscopic standards (23), the presence of unidentified contaminants is difficult to exclude rigorously. In order to establish that the binding observed in the nuclear fraction was not due to the presence of microsomal elements, a homogenate of DRG was prepared and divided into two portions.…”

Section: Identification Of Nuclear Structures As the Site Of Triton-mentioning

confidence: 99%

See 1 more Smart Citation

Nerve growth factor receptors: identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons.

Andres¹,

Jeng²,

Bradshaw³

1977

Proc. Natl. Acad. Sci. U.S.A.

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Two classes of receptors for '25I-labeled nerve growth factor in chick embryonic dorsal root neurons have been observed. One type is associated with the plasma membrane (or microsomal fraction) and can be completely solubilized by Triton X-100. These receptors display the nonsaturable binding isotherms and curvilinear Scatchard plots previously reported for nerve growth factor receptors in whole cells. The second class of binding sites is located in the nucleus, firmly bound to chromatin. These receptors are not solubilized by detergent, show saturable binding, and yield linear Scatchard plots of the type associated with a single class of binding sites of high affinity. The presence of the two receptor types suggests a bimodal mechanism of action for nerve growth factor. Nerve growth factor (NGF) is an insulin-like protein (1) that induces the morphologic and metabolic differentiation of sympathetic and sensory neurons in vertebrates (2). However, the responsiveness of these two tissues is quite different. Sensory neurons bind and are stimulated by NGF only during a relatively narrow period of embryonic development (2-4). In contrast, the sympathetic cells retain sensitivity, even in adult animals (5), although the nature of the response changes during different stages of development.The types of cellular processes effected by NGF are also variable. With low levels of hormone (about 1-10 ng), both sensory and sympathetic neurons produce the characteristic neurite outgrowth with accompanying general stimulation of anabolic metabolism and neurotubule polymerization (2, 6).At higher concentrations (about 1-10,tg), the specific induction of enzymes involved in nonadrenergic neurotransmitter synthesis is observed (7) as well as such effects as regeneration of adrenergic fibers in brain (8), inhibition of the biosynthesis of mucopolysaccharide in chrondrocytes (9), and stimulation of the temporal conversion of cell surface adhesive specificity in embryonic optic tectal cells (10).The responses associated with the low concentrations of NGF appear to be mediated through receptors bound to cell surface membranes. NGF covalently linked to Sepharose beads under conditions that prevent the release of soluble NGF produces a stimulation of neurite outgrowth from embryonic dorsal root sensory ganglia indistinguishable morphologically from that of the unbound hormone (11). By using 125I-labeled NGF (125I-NGF), these receptors have been shown to possess an affinity of approximately 1010 liters/mol (12-14), corresponding to the concentration range required for activity. However, the specific binding is a nonsaturable process with lower affinity (about 2 X 107 liters/mol) of binding sites at higher concentrations of labeled NGF (12). The range of affinities observed results from negatively cooperative interactions among a single class of receptor molecules, analogous to that observed for in- sulin receptors (15), rather than from receptor heterogeneity.Recently, it has also been demonstrated that NGF can be taken up by th...

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Section: Methodsmentioning

confidence: 99%

Section: Identification Of Nuclear Structures As the Site Of Triton-mentioning

confidence: 99%

Nerve growth factor receptors: identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons.

Andres¹,

Jeng²,

Bradshaw³

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Purity was assessed by phase-contrast microscopy. This method of neuronal nuclei isolation yields a 90 -95% pure preparation (12).…”

Section: Methodsmentioning

confidence: 99%

Effect of Moderate Hypocapnic Ventilation on Nuclear DNA Fragmentation and Energy Metabolism in the Cerebral Cortex of Newborn Piglets

et al. 2001

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Previous studies have shown that severe hypocapnic ventilation [arterial carbon dioxide partial pressure (PaCO 2 ) 7-10 mm Hg] in newborn animals results in decreased cerebral blood flow and decreased tissue oxidative metabolism. The present study tests the hypothesis that moderate hypocapnic ventilation (PaCO 2 20 mm Hg) will result in decreased cerebral oxidative metabolism and nuclear DNA fragmentation in the cerebral cortex of normoxemic newborn piglets. Studies were performed in 10 anesthetized newborn piglets. The animals were ventilated for 1 h to achieve a PaCO 2 of 20 mm Hg in the hypocapnic (H) group (n ϭ 5) and a PaCO 2 of 40 mm Hg in the normocapnic, control (C) group (n ϭ 5). Tissue oxidative metabolism, reflecting tissue oxygenation, was documented biochemically by measuring tissue ATP and phosphocreatine (PCr) levels. Cerebral cortical nuclei were purified, nuclear DNA was isolated, and DNA content was determined. DNA samples were separated, stained, and compared with a standard DNA ladder. Tissue PCr levels were significantly lower in the H group than the C group (2.32 Ϯ 0.66 versus 3.73 Ϯ 0.32 mol/g brain, p Ͻ 0.05), but ATP levels were preserved. Unlike C samples, H samples displayed a smear pattern of small molecular weight fragments between 100 and 12,000 bp. The density of DNA fragments was eight times higher in the H group than the C group, and DNA fragmentation varied inversely with levels of PCr (r ϭ 0.93). These data demonstrate that moderate hypocapnia of 1 h duration results in decreased oxidative metabolism that is associated with DNA fragmentation in the cerebral cortex of newborn piglets. We speculate that hypocapnia-induced hypoxia results in increased intranuclear Ca 2ϩ flux, which causes protease and endonuclease activation, DNA fragmentation, and periventricular leukomalacia in newborn infants. Preterm and ill term infants are at risk for brain injury, subsequent neurodevelopmental delay, and CP, partially because of alterations in CBF (1). In neonates, white matter ischemia often results in ultrasonographic evidence of PVL, which has been associated with the development of CP in human infants (2).Brain ischemia has been associated with hypocapnia owing to the effect of pH and CO 2 on cerebral vascular tone (3). In multiple studies both the degree and duration of hypocapnia have been associated with an increased incidence of PVL and CP in preterm infants (4 -6). In one study of 251 infants Ͻ34 wk gestation, 50% of ventilated preterm infants with a PaCO 2 Ͻ17 mm Hg at least once during the first 3 d of life, and 30% of those with PaCO 2 of 7-20 mm Hg, developed CP or PVL or both (6). In a study of 26 ventilated preterm infants Ͻ1500 g, 23% of infants with PaCO 2 values Ͻ20 mm Hg during the first 24 h of life developed cystic PVL compared with 6% of infants who had normal PaCO 2 values during the same period (7). In another study of 103 preterm infants, the duration of hypocapnia (defined as a PaCO 2 Յ30 mm Hg) during the first 72 h of life was an independent predictor of PVL and ...

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“…Neuronal chromatin ternplated transcription is much less restricted than that of oligodendro-and microglia [ 1,2] . These differences in chromatin template restriction are connected with differences in chromatin composition [3,4] and structure [S,lO] and may be related to different enzymatic modifications of chromosomal proteins such as the previously described higher phosphorylation of neuronal nonhistone proteins [4].…”

Section: Introductionmentioning

confidence: 99%

A comparative study of histone acetylation in neuronal and glial nuclei enriched rat brain fractions

Fleischer-Lambropoulos

1975

FEBS Letters

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Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei

Cited by 104 publications

References 46 publications

Nerve growth factor receptors: identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons.

Nerve growth factor receptors: identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons.

Effect of Moderate Hypocapnic Ventilation on Nuclear DNA Fragmentation and Energy Metabolism in the Cerebral Cortex of Newborn Piglets

A comparative study of histone acetylation in neuronal and glial nuclei enriched rat brain fractions

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