The synthesis of ribonucleic acid in vivo in the nuclei of rat brain fractionated by zonal centrifugation

1. Radioactive orotic acid, uridine and adenosine were administered to rats by intracisternal injection. The effects of the size of the dose, the specific radioactivity and time on the incorporation into the RNA of unfractionated nuclei of brain tissue were examined to establish appropriate conditions for studies of the relative activities in vivo of the various sorts of brain nuclei fractionated by zonal centrifugation. Uridine is incorporated more efficiently than either orotic acid or adenosine. 2. With [3H]uridine as precursor the astrocytes in zone (II) contain the highest radioactivity except at the beginning of the experiment when the neuronal nuclei of zone (I) are more highly labelled. This fraction utilizes [14C]orotic acid more readily than the nuclei of zone (II). The astrocytic nuclei of zone (III) show a general resemblance to those of zone (II). Considerable differences between the incorporations into the two types of oligodendrocyte nuclei in zones (IV) and (V) are observed. 3. The relative synthetic activities of the major types of brain nuclei in vitro and in vivo are discussed.

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Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

Biochemical Journal ◽

10.1042/bj1050779 ◽

1967 ◽

Vol 105 (2) ◽

pp. 779-782 ◽

Cited By ~ 131

Author(s):

F. Stirpe ◽

L. Fiume

Keyword(s):

Rna Polymerase ◽

Ammonium Sulphate ◽

Ribonucleic Acid ◽

Mouse Liver ◽

Orotic Acid ◽

Acid Synthesis ◽

Liver Necrosis ◽

Liver Nuclei

1. Injection of α-amanitin to mice causes a decreased incorporation of [6−14C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn2+ and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with α-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg2+-activated RNA polymerase is only slightly affected by α-amanitin either administered to mice or added in vitro.

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The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation

Biochemical Journal ◽

10.1042/bj1360685 ◽

1973 ◽

Vol 136 (3) ◽

pp. 685-695 ◽

Cited By ~ 25

Author(s):

M. A. Stambolova ◽

D. Cox ◽

A. P. Mathias

Keyword(s):

Rat Brain ◽

Dna Polymerase ◽

Deoxyribonucleic Acid ◽

Specific Radioactivity ◽

Brain Nuclei ◽

Neuronal Nuclei ◽

Adult Rat ◽

Infant Rats ◽

Discontinuous Sucrose Gradient ◽

Adult Rat Brain

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg2+, glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [3H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [3H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).

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Effect of α-amanitin on ribonucleic acid polymerase II of rat brain nuclei and on retention of avoidance conditioning

Biochemical Journal ◽

10.1042/bj1251087 ◽

1971 ◽

Vol 125 (4) ◽

pp. 1087-1090 ◽

Cited By ~ 16

Author(s):

N. Montanaro ◽

F. Novello ◽

F. Stirpe

Keyword(s):

Rna Polymerase ◽

Rat Brain ◽

Rna Polymerase Ii ◽

Ribonucleic Acid ◽

Avoidance Conditioning ◽

Avoidance Task ◽

Intracerebral Injection ◽

Brain Nuclei ◽

Impaired Performance

1. α-Amanitin inhibits in vitro the activity of RNA polymerase II of rat brain nuclei. 2. The toxin does not pass the blood–brain barrier, but when injected intracerebrally is highly toxic for rats, and causes inhibition of RNA polymerase II of isolated brain nuclei. 3. Intracerebral injection of α-amanitin 6h before training to a passive avoidance task is followed by impaired performance of rats on retesting after 7 days, without affecting performance on retesting immediately after training.

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Active-site-directed irreversible inhibition of rat brain 4-aminobutyrate aminotransferase by ethanolamine O-sulphate in vitro and in vivo

Biochemical Journal ◽

10.1042/bj1300569 ◽

1972 ◽

Vol 130 (2) ◽

pp. 569-573 ◽

Cited By ~ 153

Author(s):

L. J. Fowler ◽

R. A. John

Keyword(s):

Rat Brain ◽

Dependent Manner ◽

Aminobutyrate Aminotransferase ◽

First Order ◽

Irreversible Inhibition ◽

First Order Kinetics ◽

Intracisternal Injection ◽

Pseudo First Order

1. Partially purified preparations of rat brain 4-aminobutyrate aminotransferase were inhibited in a time-dependent manner by ethanolamine O-sulphate. The inhibition was not reversed by dialysis. 2. The inhibitor formed an initial reversible complex with the enzyme (Ki=4.4×10−4m) and the rate of inactivation followed pseudo-first-order kinetics (k=7.15×10−4s−1). The inclusion of 4-aminobutyrate markedly slowed the rate of inactivation. 3. Ethanolamine O-sulphate did not inhibit glutamate decarboxylase, alanine aminotransferase or aspartate aminotransferase. 4. Intracisternal injection of ethanolamine O-sulphate into rats led to rapid inactivation of 4-aminobutyrate aminotransferase in vivo.

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Radiolabeling of Fibrinogen Using the Iodogen Technique

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653425 ◽

1981 ◽

Vol 46 (03) ◽

pp. 593-596 ◽

Cited By ~ 52

Author(s):

Linda C Knight ◽

Andrei Z Budzynski ◽

Stephanie A Olexa

Keyword(s):

Specific Radioactivity ◽

Oxidizing Agent ◽

Iodine Monochloride ◽

Human Fibrinogen

SummaryThe properties of human fibrinogen labeled with 125-Iodine using Iodogen (1, 3, 4, 6-tetrachloro-3α, 6α-diphenylglycoluril) as an oxidizing agent were compared with those of an iodine monochloride labeled counterpart. It was found that thrombin clottability, binding to staphylococci, the relative specific radioactivity of the Aα, Bβ, and γ chains and in vivo clearance from plasma in rabbits were the same in these two labeled fibrinogen preparations. Labeling efficiency was higher when iodogen was used. It is concluded that human fibrinogen labeled with radioiodine using the Iodogen technique is suitable for studies in vitro and in vivo.

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CONTRIBUTION TO THE EXISTENCE OF REGULATORY MECHANISMS AT THE ADRENAL LEVEL

Acta Endocrinologica ◽

10.1530/acta.0.0700741 ◽

1972 ◽

Vol 70 (4) ◽

pp. 741-757

Author(s):

Otto Linèt

Keyword(s):

Orotic Acid ◽

Adrenal Glands ◽

Actinomycin D ◽

Delay Period ◽

Regulatory Mechanisms ◽

Leucine Incorporation ◽

Total Period ◽

Free Media

ABSTRACT Rat adrenal glands atrophied by the administration of cortisol acetate in vivo were used as a model for the study of early metabolic processes occurring in vitro. Atrophied adrenals incubated in the presence of 14C-leucine incorporated subnormal quantities of this amino acid per mg of protein for the first 120 min. When the incubation lasted for a total period of 180 or 240 min a supranormal rise in the 14C-leucine incorporation was observed. Similar changes occurred with some delay with regard to corticosterone production as expressed per 100 mg of tissue. No differences in 14C-leucine incorporation were observed between the control and atrophied adrenals in vivo. Homogenates from atrophied glands incorporated 14C-leucine to a greater extent than the control homogenates. The in vitro incorporation of 14C-orotic acid into the RNA was also higher in atrophied adrenals. The in vitro use of actinomycin D, cycloheximide and amphenone indicated that corticosterone production depended on the incorporation of 14C-leucine. The addition of cortisol to the incubation media markedly decreased the enhancement of 14C-lysine incorporation into the protein of atrophied adrenals. These, as well as additional results suggest rebound phenomena: once atrophic adrenals are transferred to cortisol-free media, reparative processes begin after a delay period. Such phenomena seem to be mediated by regulatory mechanisms at the adrenal level.

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Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

Biochemical Journal ◽

10.1042/bj20020123 ◽

2002 ◽

Vol 364 (2) ◽

pp. 343-347 ◽

Cited By ~ 51

Author(s):

Gareth J.O. EVANS ◽

Alan MORGAN

Keyword(s):

Rat Brain ◽

Direct Interaction ◽

Secretory Vesicle ◽

Brain Membrane ◽

Synaptotagmin I ◽

Phosphorylated Form ◽

Order Of Magnitude ◽

And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

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