Ligation and synthesis of chromatin deoxyribonucleic acid in vitro in neuronal, glial and liver nuclei isolated from adult guinea pig

Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.     

Decreased ribonucleic acid synthesis in isolated rat liver nuclei during starvation

1. Isolated nuclei from starved rats showed a lowered incorporation of [(14)C]UMP into RNA. 2. The Mg(2+)-dependent incorporation was decreased by 30% after 1 day of starvation, but incorporation in the presence of Mn(2+) and ammonium sulphate decreased only after longer periods of starvation. 3. RNA synthesis by nuclei in the presence of excess of added RNA polymerase was unchanged after 1 day of starvation and was inhibited by 20% after 4 days. 4. The capacity of nuclei to bind actinomycin D was unchanged after 1 day and was decreased by 20% after 4 days of starvation.     

Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury

Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability.     

Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Studies on ribonucleic acid metabolism using nuclear columns. Release of rapidly labeled RNA from rat liver nuclei.

A method is described to study the effect of successively changing incubation conditions on the release of rapidly labeled RNA from isolated nuclei. Nuclear columns containing immobilized rat liver nuclei isolated after in vivo application of labeled orotic acid are perfused with different non-radioactive media. Within the course of one perfusion, the rate of RNA release can be repeatedly altered by variation of temperature, acidity and concentrations of nucleoside triphosphates, complexing agents, sodium chloride and manganese chloride. RNA release can be started and stopped, indicating that the reaction does not result from damage to nuclei. During 60 min perfusion the same product, labeled ribonucleoprotein (sigma = 1.43 g/cm3 in CsCl), is released. High release rates depend on the ratio of nucleoside triphosphate to divalent cation concentration, not on the concentration of the agents per se. Ribonucleoside and deoxyribonucleoside triphosphates exert the same effect as ATP. The SH reagents iodoacetamide and iodoacetate only slightly affect the ATP-induced reaction. In contrast, p-chloromercuribenzoate, after an initial stimulation, causes inhibition of RNA release.     

Messenger ribonucleic acid of cerebral nuclei

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.     

Age-associated changes in initiation of ribonucleic acid synthesis in isolated rat liver nuclei.

Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.     

Ribonucleic acid synthesis in isolated rat liver nuclei under conditions of ribonucleic acid processing and transport.

A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.