scholarly journals A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor

1969 ◽  
Vol 111 (4) ◽  
pp. 583-591 ◽  
Author(s):  
A P Mathias ◽  
D. Ridge ◽  
N. St G. Trezona
Keyword(s):  
Molecular Weight ◽  
Ribosomal Rna ◽  
Base Composition ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Density Gradients ◽  
Avian Erythrocytes ◽  
Nuclear Rna ◽  
Zonal Rotor

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [32P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35μ3 and are isopycnic with sucrose of density 1·2871 (measured at 20°). Maturation of the nuclei causes them to shrink to a volume of 25μ3 and the density to increase to 1·2944.

Metabolism of high molecular weight, polydisperse, rapidly labeled nuclear RNA in rat liver

1968 ◽  
Vol 46 (12) ◽  
pp. 1497-1505 ◽  
Author(s):  
Maurice Brossard ◽  
Louis Nicole
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Rat Liver ◽  
Ribosomal Rna ◽  
Base Composition ◽  
Orotic Acid ◽  
Chromosomal Origin ◽  
Base Analysis ◽  
Nuclear Rna ◽  
Kinetics Of

Studies of the metabolism of rat liver RNA showed the existence of two species of rapidly labeled nuclear RNA: a 45 S preribosomal type of nucleolar origin, and a 6–50 S polydisperse RNA of chromosomal origin. The kinetics of labeling with orotic acid-14C and the nature of the latter RNA have been investigated. The following findings are reported, (1) This RNA is composed of at least four main classes of RNA having sedimentation coefficients of approximately 45, 35, 24, and 18 S. (2) Except for the 18 S class which seems to be an end product, the three other classes have a rapid turnover and do not appear to leave the nucleus. (3) Base analysis after 32P incorporation indicates that these four classes of RNA have a similar base composition with a G+C/A + U ratio in the range of 0.98–1.07, which resembles DNA more closely than ribosomal RNA. (4) The 6–50 S polydisperse RNA has a different metabolism than that of the 45 S preribosomal type and there is no precursor-to-product relationship between these two species of RNA.

Mise en évidence d'ADN extrachromosomique chez Rhizobium meliloti

1978 ◽  
Vol 24 (8) ◽  
pp. 960-966 ◽  
Author(s):  
M. Bechet ◽  
J. B. Guillaume
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Base Composition ◽  
Density Gradient Centrifugation ◽  
Rhizobium Meliloti ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Sedimentation Analysis ◽  
Extrachromosomal Dna ◽  
Very High

Seven effective (nitrogen-fixing) strains of Rhizobium meliloti have been studied. By sedimentation analysis of their alkaline lysates in alkaline sucrose gradients, a plasmid was found in four strains. In a strain (2011 str 3) which gave no result with this method, supercoiled DNA was detected by CsCl-dye buoyant density gradient centrifugation. That result was confirmed by analytical Cs2SO4–Ag+ density gradients, which showed a heterogeneity in the average base composition of the DNA extracted from three strains, including the 2011 str 3 strain. Two of those last strains seemed to contain an extrachromosomal DNA of very high molecular weight.

scholarly journals Oestradiol inhibits collagen breakdown in the involuting rat uterus

1972 ◽  
Vol 127 (4) ◽  
pp. 705-713 ◽  
Author(s):  
Janet N. Ryan ◽  
J. Frederick Woessner
Keyword(s):  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Post Partum ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Rat Uterus ◽  
Oestradiol Treatment ◽  
Cathepsin D Activity ◽  
Stimulation Of

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100μg of oestradiol-17β/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[14C]-proline by the administration of [14C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[14C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [14C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.

DNA Synthesis in Isolated Yeast Mitochondria

1974 ◽  
Vol 52 (11) ◽  
pp. 941-949 ◽  
Author(s):  
L. Zeman ◽  
C. V. Lusena
Keyword(s):  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Base Composition ◽  
Nuclear Dna ◽  
Sedimentation Velocity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Denatured State ◽  
Saccharomyces Cerevisiae Mitochondria

Isolated yeast (Saccharomyces cerevisiae) mitochondria incorporate radioactive precursors into mitochondrial DNA. This in vitro labelled DNA was characterized by isopycnic and sedimentation velocity centrifugation both in the native and denatured state. The profiles of isopycnic CsCl gradients obtained by centrifugation in a fixed-angle rotor are skewed toward high density. The skew is neither due to the presence of in vitro labelled nuclear DNA nor due to random breaks in mitochondrial DNA which would reveal, then, its heterogeneity in base composition. The in vitro labelled DNA is reproducibly recovered as a class of molecules sedimenting at about 5–8 S, indicating a molecular weight of 1 × 105 – 4 × 105 daltons, while the smallest in vivo labelled fragments sediment at about 13–14 S, corresponding to 1.6 × 106 – 2.0 × 106 daltons. After denaturation, the in vitro labelled DNA molecules sediment at about 2–5 S, corresponding to a single-strand molecular weight of 1 × 104 – 7 × 104 daltons, which is about one hundred times less than the observed size of the denatured in vivo labelled molecules.

scholarly journals SYNTHESIS OF CHLOROPLAST DNA IN ISOLATED CHLOROPLASTS

1968 ◽  
Vol 38 (1) ◽  
pp. 151-157 ◽  
Author(s):  
N. Steele Scott ◽  
Vinod C. Shah ◽  
Robert M. Smillie
Keyword(s):  
Molecular Weight ◽  
Chloroplast Dna ◽  
Euglena Gracilis ◽  
Bacterial Contamination ◽  
Actinomycin D ◽  
Density Gradient Centrifugation ◽  
Tritiated Thymidine ◽  
Gradient Centrifugation ◽  
Acid Stable ◽  
Isolated Chloroplasts

Chloroplasts isolated from Euglena gracilis incorporated both tritiated thymidine 5'-triphosphate and tritiated deoxyadenosine 5'-triphosphate into an acid-stable fraction. The incorporation was dependent on the presence of all four deoxynucleoside triphosphates and was sensitive to treatment with deoxyribonuclease and actinomycin D. It was demonstrated that bacterial contamination could not account for the incorporation of label. Extraction of DNA from the chloroplasts and subsequent density gradient centrifugation of the DNA in CsCl2 showed that the incorporation was into chloroplast DNA (ρ = 1.686) of high molecular weight.

Intranuclear Accumulation of Rna Resembling the Smaller Ribosomal Rna Component

1970 ◽  
Vol 6 (1) ◽  
pp. 53-72
Author(s):  
M. E. BRAMWELL
Keyword(s):  
Ribosomal Rna ◽  
Base Composition ◽  
Actinomycin D ◽  
Total Radioactivity ◽  
16S Ribosomal Rna ◽  
Low Concentrations ◽  
Rapid Accumulation ◽  
Nuclear Rna ◽  
Step Down ◽  
Nucleolar Rna

A study was made of the nuclear RNA in HeLa cells with particular reference to the rapidly labelled fractions. It was found that if cells were incubated at a high density, that is, under ‘step-down’ conditions, there was a rapid accumulation of RNA in the nucleus. The fraction of the nuclear RNA which includes rapidly labelled RNA and which binds tightly to columns of methylated albumin on kieselguhr increased in amount and reached levels which permitted enough of the material to be isolated for direct measurement of its base composition. This was found to be very similar to that of 16s ribosomal RNA. When cells growing logarithmically were treated with low concentrations of actinomycin D and then incubated in the presence of [3H]uridine it was found that an RNA fraction which bound tightly to methylated albumin on kieselguhr again accumulated in the nucleus. This fraction resembled that which accumulated under ‘step-down’ conditions. It contained over 85% of the total radioactivity in the nuclear RNA and again had a base composition very similar to 16s ribosomal RNA. Since nucleolar RNA synthesis was inhibited by the concentrations of actinomycin D used, it appeared that an RNA closely resembling 16s ribosomal RNA was synthesized outside the nucleolus. Sedimentation patterns on sucrose density gradients and thermal denaturation profiles lent support to the view that the RNA which binds tightly to columns of methylated albumin on kieselguhr probably represents ‘nascent’ 16s ribosomal RNA.

scholarly journals Evidence that platelet density depends on the alpha-granule content in platelets

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 482-485 ◽  
Author(s):  
BA van Oost ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Density Distribution ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Platelet Rich Plasma ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Adenosine Diphosphate ◽  
Density Gradients

Abstract The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.

scholarly journals Separation of Degranulated Platelets from Normal Platelets

1977 ◽  
Author(s):  
P. Cieslar ◽  
J.P. Greenberg ◽  
M.A. Packham ◽  
R.L. Kinlough-Rathbone ◽  
J.F. Mustard
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Adenine Nucleotide ◽  
Thrombus Formation ◽  
Gradient Centrifugation ◽  
Rabbit Platelets ◽  
Fraction I

Platelets degranulated by thrombin (TDP) can be recovered, are effective in hemostasis and survive normally upon infusion into rabbits. Two approaches to determine whether platelets have been degranulated in vivo are: (1) measurement of circulating released materials; (2) detection of circulating degranulated platelets. We have used arabino-galactan (Stractan II) density gradient centrifugation to separate normal and degranulated platelets. The following distribution was obtained with washed rabbit platelets.The serotonin, PF4 and adenine nucleotide contents of the TDP were less than those of normal platelets and the TDP in fraction I had the lowest amounts. When TDP were labeled with 51cr and mixed with equal numbers of normal platelets, 85% of the platelets in fraction I were found to be TDP. 51Cr-TDP were injected into normal rabbits and reharvested after 18 hours. The greatest proportion of TDP was isolated in fraction I. Thus this method may make it possible to separate platelets that have lost their granule contents during participation in reversible thrombus formation in vivo.(* Visiting Fellow from the Faculty of Medicine, Charles University, Prague, Czechoslovakia.)

scholarly journals MITOCHONDRIAL RNA FROM CULTURED ANIMAL CELLS

1970 ◽  
Vol 46 (2) ◽  
pp. 245-251 ◽  
Author(s):  
Bland S. Montenecourt ◽  
Margaret E. Langsam ◽  
Donald T. Dubin
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cell Lines ◽  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Base Composition ◽  
Size Class ◽  
Mitochondrial Rna ◽  
Animal Cells ◽  
Molecular Weights

Discrete RNA fractions sedimenting slightly slower than 18s ribosomal RNA have been found in mitochondrial preparations from both hamster (BHK-21) and mouse (L-929) cells. This RNA could be separated into two components, present in approximately equimolar amounts, by prolonged zonal centrifugation or acrylamide gel electrophoresis. The hamster components had sedimentation constants averaging 16.8 and 13.4, and molecular weights (estimated by gel electrophoresis) averaging 0.74 and 0.42 x 106 daltons. Mixed labeling experiments showed that the mouse components sedimented and electrophoresed 3–6% more slowly than the corresponding hamster components. The RNA from both cell lines resembled mitochondrial ribosomal RNA from yeast and Neurospora in being GC poor, and in addition the larger and smaller components resembled each other in base composition. These results, taken with those of other recent studies, are compatible with the idea that our high molecular weight mitochondrial RNA is ribosomal; such RNA would then constitute a uniquely small size-class of ribosomal RNA.

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