scholarly journals STUDIES ON ISOLATED NUCLEI

1963 ◽  
Vol 18 (2) ◽  
pp. 293-312 ◽  
Author(s):  
Rachele Maggio ◽  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Nucleotide Composition ◽  
Gradient System ◽  
Variable Amount ◽  
Cell Nuclei ◽  
Pig Liver ◽  
Isolated Nuclei ◽  
Guinea Pig Liver ◽  
Rna And Dna ◽  
Degree Of Fragmentation

This paper describes the subfractionation of nuclei isolated from guinea pig liver by the procedure presented in the first article of the series (8). Centrifugation in a density gradient system of nuclear fractions disrupted by sonication permits the isolation of the following subfractions: (a) a nucleolar subfraction which consists mainly of nucleoli surrounded by a variable amount of nucleolus-associated chromatin and contaminated by chromatin blocks derived primarily from von Kupffer cell nuclei; (b) and (c), two nucleoplasmic subfractions (I and II) which consist mainly of chromatin threads in a coarser (I) or finer (II) degree of fragmentation. The protein, RNA, and DNA content of these subfractions was determined, and their RNA's characterized in terms of NaCl-solubility, nucleotide composition, and in vivo nucleotide turnover, using inorganic 32P as a marker. The results indicate that there are at least three types of RNA in the nucleus (one in the nucleolus and two in the nucleoplasm or chromatin), which differ from one another in NaCl-solubility, nucleotide composition, turnover, and possibly sequence. Possible relations among these RNA's and those of the cytoplasm are discussed.

scholarly journals The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei

1969 ◽  
Vol 112 (1) ◽  
pp. 71-79 ◽  
Author(s):  
J. W. Watts
Keyword(s):  
Hela Cell ◽  
Incubation Medium ◽  
Sedimentation Coefficient ◽  
Cell Nuclei ◽  
Isolated Nuclei ◽  
Bivalent Cations ◽  
Low Concentrations ◽  
Nuclear Rna ◽  
And Diffusion

1. The loss of nucleic acids and protein from isolated HeLa-cell nuclei was studied. During 4hr. incubation at 37° DNA was conserved, but appreciable amounts of RNA and protein were lost. 2. Two classes of nuclear RNA were distinguished: at least 75% of the RNA was lost from the nuclei relatively slowly through degradation to acid-soluble fragments; the rest of the RNA was lost much more rapidly, not only through degradation to acid-soluble fragments but also through diffusion of RNA out of the nuclei into the incubation medium. 3. The RNA that was preferentially lost was the fraction of nuclear RNA that was rapidly labelled when intact HeLa cells were grown in a medium containing radioactive precursors of RNA. 4. The RNA appearing in the incubation medium was apparently partially degraded and had a sedimentation coefficient of about that of transfer RNA. 5. Both the degradation of RNA and the loss of RNA from the nuclei were sensitive to bivalent cations. Low concentrations of Mg2+ and Mn2+ greatly increased the rate of degradation of the rapidly labelled RNA to acid-soluble fragments, and produced a corresponding decrease in the amount of RNA diffusing into the medium. At higher concentrations they suppressed both degradation and diffusion of RNA. The cations Ca2+, Cu2+, Zn2+ and Ni2+ all progressively inhibited both forms of loss of RNA. 6. Salts of univalent cations produced appreciable effects only at ionic strengths of about 0·2, when degradation to acid-soluble fragments was preferentially inhibited. 7. Both ADP and ATP inhibited loss of RNA at about 30mm. 8. It was concluded that the diffusion of rapidly labelled RNA out of the isolated nuclei was not related to the movement of RNA from nucleus to cytoplasm in vivo, but reflected the ease with which the rapidly labelled RNA detached from the chromatin and the permeability of the membranes of isolated nuclei.

scholarly journals Lipid synthesis in vivo by tissues of the maternal and foetal guinea pig

1976 ◽  
Vol 154 (1) ◽  
pp. 159-161 ◽  
Author(s):  
C T Jones ◽  
W Firmin
Keyword(s):  
Fatty Acids ◽  
Adipose Tissue ◽  
Guinea Pig ◽  
Lipid Synthesis ◽  
Short Chain Fatty Acids ◽  
Adipose Tissues ◽  
Pig Liver ◽  
Foetal Liver ◽  
Guinea Pig Liver

The rate of lipid biosynthesis in vivo was determined in pregnant guinea pigs after maternal and foetal injections of 3H2O. Synthesis in the maternal tissues was low and in the foetal liver and adipose tissues relatively high. In the foetal liver it reached a peak at about two-thirds of gestation, whereas that in the foetal adipose tissue occurred later. These results were used to support the view that lipid synthesis in the foetal guinea-pig liver at two-thirds of gestation is largely from short-chain fatty acids, whereas in foetal adipose tissue glucose is probably the major substrate.

scholarly journals The availability of carnitine acetyltransferase in mitochondria from guinea-pig liver and other tissues

1968 ◽  
Vol 110 (4) ◽  
pp. 739-746 ◽  
Author(s):  
P. J. Barker ◽  
N. J. Fincham ◽  
D C Hardwick
Keyword(s):  
Guinea Pig ◽  
Glutamate Dehydrogenase ◽  
Freeze Drying ◽  
Enzymic Activity ◽  
Carnitine Acetyltransferase ◽  
Cell Cytoplasm ◽  
Pig Liver ◽  
Triton X ◽  
Guinea Pig Liver

The carnitine acetyltransferase and glutamate dehydrogenase activities of guinea-pig liver and other tissues were estimated. Both enzymes are wholly mitochondrial, and can only be fully observed after disruption of the mitochondrion. Triton X-100 (0·1%) or freeze-drying revealed more activity than other methods tried. In mitochondria prepared and suspended in 0·25m-sucrose and in cell cytoplasm only small fractions of the total enzymic activity could be observed in guinea-pig liver: on average 7·5% of carnitine acetyltransferase and 5·5% of glutamate dehydrogenase. It is concluded that, in liver or mammary gland of goat, guinea pig or rat, little or no carnitine acetyltransferase is available in vivo to acetyl-CoA outside the mitochondrion.

scholarly journals Effect of physiological reducing compounds on the inactivation of tyrosine aminotransferase from guinea-pig liver*

1982 ◽  
Vol 205 (2) ◽  
pp. 265-269 ◽  
Author(s):  
D Di Cola ◽  
G Federici
Keyword(s):  
Guinea Pig ◽  
Reduced Glutathione ◽  
Tyrosine Aminotransferase ◽  
Enzyme Inactivation ◽  
Inactivation Rate ◽  
Physiological Concentration ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Guinea Pig Liver

1. Tyrosine aminotransferase from guinea-pig liver is inactivated at neutral pH by a factor localized in the microsomal fraction. The inactivation, independent of exogenous L-cysteine, is rapidly reversed by addition of dithiothreitol. 2. The effects of physiological reducing agents on the enzyme inactivation were investigated. L-Cysteine and L-cysteamine enhance the inactivation rate of the enzyme in the presence of microsomal membranes, and also they are able to bring about the loss in enzyme activity independently of microsomal action. Reduced glutathione, at physiological concentration, and NADPH decrease the inactivation rate. Other physiological reducing compounds, as well as oxidized glutathione and NADP+, are without effect. 3. Neither reduced glutathione nor NADPH, unlike dithiothreitol and mercaptoethanol, is able to restore the activity of partially inactivated tyrosine aminotransferase. 4. It is proposed that the intracellular concentration of reduced glutathione might modulate the rate of inactivation of the enzyme in vivo.

In vitro and in vivo studies on the metabolism of estrogens and their sulfates in guinea pigs

1977 ◽  
Vol 55 (4) ◽  
pp. 390-397 ◽  
Author(s):  
R. Hobkirk ◽  
D. J. Freeman ◽  
P. R. C. Harvey ◽  
Mona Nilsen ◽  
Barbara Jennings
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Mature Male ◽  
In Vivo Studies ◽  
Pig Liver ◽  
Male And Female ◽  
Estradiol 17Β ◽  
Guinea Pig Liver

Labelled estradiol-17β(E2) or estrone (E1), when incubated with guinea pig liver slices, is metabolized by two main pathways. Part of each substrate is converted to estrone-3-glucuronide and estradiol-3-glucuronide. A further part of each is metabolized to estradiol-3-sulfate (E23S) and estrone-3-sulfate (E13S), which are interconverted. The latter conjugate appears to be the substrate for a 16α-hydroxylase forming 16α-hydroxyestrone-3-sulfate (16αOHE13S). This, in turn, is further sulfurylated to yield 16α-hydroxyestrone-3,16-disulfate, accompanied by estriol-3,16-disulfate. A relatively small amount of tentatively identified '6-hydroxyestrone disulfate' accompanies these other two diconjugates. The guinea pig liver system suggests itself as a useful and relatively simple model for further study of 16α-hydroxylation of E13S. The use of the latter as a natural substrate in the system in vitro is supported by our observation that E13S and E23S are present in liver, kidney, blood, gallbladder bile, intestine, uterus, and placenta after injection of labelled E2 into mature male and female guinea pigs. Some evidence has been obtained for the disulfate fraction (above) in liver and bile after injection of labelled E1.

scholarly journals STUDIES ON ISOLATED NUCLEI

1963 ◽  
Vol 18 (2) ◽  
pp. 267-291 ◽  
Author(s):  
Rachele Maggio ◽  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Nuclear Envelope ◽  
Base Composition ◽  
Nuclear Fraction ◽  
Pig Liver ◽  
Lower Phase ◽  
Dna Recovery ◽  
Isolated Nuclei ◽  
Nuclear Rna ◽  
Electron Microscopical ◽  
Guinea Pig Liver

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl2 followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl2. Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that ∼88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that ∼90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of ∼22.3 per cent DNA, ∼4.7 per cent RNA, and ∼73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.

Die Bedeutung der Homogenisationsart und -intensität für die Größe und Konstanz der Sauerstoffaufnahme von Meerschweinchen-Leberhomogenat / The Influence of Mode and Intensity of Homogenization on the Absolute Value and Stability of Oxygen Consumption of Guinea Pig Liver Homogenates

1977 ◽  
Vol 32 (11-12) ◽  
pp. 908-912 ◽  
Author(s):  
H. J. Schmidt ◽  
U. Schaum ◽  
J. P. Pichotka
Keyword(s):  
Oxygen Uptake ◽  
Guinea Pig ◽  
Measuring Time ◽  
Pig Liver ◽  
Absolute Value ◽  
Modified Method ◽  
Simultaneous Measurements ◽  
Liver Homogenates ◽  
The Absolute ◽  
Guinea Pig Liver

Abstract The influence of five different methods of homogenisation (1. The method according to Potter and Elvehjem, 2. A modification of this method called Potter S, 3. The method of Dounce, 4. Homogenisation by hypersonic waves and 5. Coarce-grained homogenisation with the “Mikro-fleischwolf”) on the absolute value and stability of oxygen uptake of guinea pig liver homogenates has been investigated in simultaneous measurements. All homogenates showed a characteristic fall of oxygen uptake during measuring time (3 hours). The modified method according to Potter and Elvehjem called Potter S showed reproducible results without any influence by homogenisation intensity.

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