The fractionation of nuclei from mammalian cells by zonal centrifugation

Johnston, Irving R.; Mathias, A. P.; Pennington, Frank C.; Ridge, David

doi:10.1042/bj1090127

Cited by 78 publications

(23 citation statements)

References 11 publications

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“…The second peak was composed predominantly of tetraploid parenchymal nuclei. The separations were very similar to those obtained in solutions of sucrose (Johnston et al [2]). …”

Section: Fractionation Of Rat Liver Nwleisupporting

confidence: 86%

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The uses of metrizamide in the fractionation of nuclei from brain and liver tissue by zonal centrifugation

Mathias¹,

Wynter²

1973

FEBS Letters

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“…The second peak was composed predominantly of tetraploid parenchymal nuclei. The separations were very similar to those obtained in solutions of sucrose (Johnston et al [2]). …”

Section: Fractionation Of Rat Liver Nwleisupporting

confidence: 86%

“…Nuclei were counted in a Coulter Counter Model F with 100 pm orifice. Nuclei were prepared from livers 18 of Wistar rats as described by Johnston et al [2].…”

Section: Isolation Of Nucleimentioning

confidence: 99%

The uses of metrizamide in the fractionation of nuclei from brain and liver tissue by zonal centrifugation

Mathias¹,

Wynter²

1973

FEBS Letters

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“…We have observed (38) that in rats treated with hypolipidemic drugs for 18 months there are more mitotic figures in test than in control animals, indicating an increased rate of cell death and replacement. The nuclei of a very high proportion of the hepatocytes of aged rats and mice are polyploid (74,75); polyploid nuclei divide with about the same frequency as diploid (76), and it is believed that an increase in ploidy will increase the risk of chromosome damage during mitosis. Furthermore, this theory is similar to one proposed to explain the carcinogenicity of plastic ifim implants (77).…”

Section: Development Of Hepatic Changesmentioning

confidence: 99%

Effects of phthalic acid esters on the liver and thyroid.

Hinton¹,

Mitchell²,

Mann³

et al. 1986

Environ Health Perspect

127

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The effects, over periods from 3 days to 9 months of administration, of diets containing di-2-ethylhexyl phthalate are very similar to those observed in rats administered diets containing hypolipidemic drugs such as clofibrate. Changes occur in a characteristic order commencing with alterations in the distribution of lipid within the liver, quickly followed by proliferation of hepatic peroxisomes and induction of the specialized P450 isoenzyme(s) catalyzing w oxidation of fatty acids. There follows a phase of mild liver damage indicated by induction of glucose-6-phosphatase activity and a loss of glycogen, eventually leading to the formation of enlarged lysosomes through autophagy and the accumulation of lipofuscin. Associated changes are found in the kidney and thyroid. The renal changes are limited to the proximal convoluted tubules and are generally similar to changes found in the liver. The effects on the thyroid are more marked. Although the levels of thyroxine in plasma fall to about half normal values, serum triiodothyronine remains close to normal values while the appearance of the thyroid varies, very marked hyperactivity being noted 7 days after commencement of treatment, this is less marked at 14 days, but even after 9 months treatment there is clear cut evidence for hyperactivity with colloid changes which indicate this has persisted for some time. Straight chain analogs of di-2-ethylhexyl phthalate, di-n-hexyl phthalate and di-n-oxtyl phthalate differ entirely in their short-term effects on the liver and kidney but have similar effects on the thyroid.The short-term in vivo hepatic effects of the three phthalate esters can be reproduced in hepatocytes in tissue culture. All three phthalate esters, as well as clofibrate, have early marked effects on the metabolism of fatty acids in isolated hepatocytes. The nature of these changes is such as to increase storage of lipid in the liver. A hypothesis is presented to explain the progress from these initial metabolic effects to the final formation of liver tumors.

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“…(1967) did not study sucrose-CaC12 nuclei . Several criticisms of utilizing the present rat liver nuclei are (a) that the cells in the liver are in different stages of the cell mitotic cycle and this might result in different nuclei (McBride and Peterson, 1970) ; (b) that liver cells contain diploid, tetraploid, and higher ploidy nuclei ; (c) that liver contains several cell types, each of which has a slightly different nucleus (Johnston et al ., 1968) ; that the nuclei could be enveloped in plasma membrane fragments, as found for avian erythrocyte nuclei (Zentgrab et al ., 1969) . In all instances in the present experiments, the nuclear (and nucleolar) preparations behaved as homogeneous, not heterogeneous, populations with respect to their electrophoretic mobilities, and the analysis of the nuclear fractions for the plasma membrane marker enzyme 5'-nucleotidase was negative.…”

Section: Discussionmentioning

confidence: 99%

Molecules at the External Nuclear Surface

Bosmann

1973

The Journal of Cell Biology

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The molecules occurring as terminal residues on the external surfaces of nuclei prepared from rat liver by either sucrose-CaC12 or citric acid methods and nucleoli derived from the sucrose-CaC1 2 nuclei were studied chemically and electrokinetically . In 0.0145 M NaCl, 4 .5 0 /,) sorbitol, and 0 .6 mM NaHCO3 with pH 7 .2 t 0 .1 at 25°C, the sucrose-CaC12 nuclei had an electrophoretic mobility of -1 .92 ,um/s/V/cm, the citric acid nuclei, -1 .63 µm/s/V/cm, and the nucleoli, -2 .53 µm/s/V/cm . The citric acid nuclei and the nucleoli contained no measurable sialic acid . The sucrose-CaC12 nuclei contained 0.7 pmol of sialic acid/mg nuclear protein ; this was essentially located in the nuclear envelope . Treatment of these nuclei with 50 µg neuraminidase/mg protein resulted in release of 0 .63 pmol of sialic acid/mg nuclear protein ; treatment with 1 % trypsin caused release of 0 .39 nmol of the sialic acid/mg nuclear protein . The pH-mobility curves for the particles indicated the sucrose-CaC12 nuclei surface had an acid-dissociable group of pK -2 .7 while the pK for the nucleoli was considerably lower . Nucleoli treated with 50 µg neuraminidase/mg particle protein had a mobility of -2 .53 Am/s/V/cm while sucrose-CaC12 nuclei similarly treated had a mobility of -1 .41 µm/s/V/cm . Hyaluronidase at 50 µg/mg protein had no effect on nucleoli mobility but decreased the sucrose-CaC12 nuclei mobility to -1 .79 M.m/s/V/cm . Trypsin at 1 % elevated the electrophoretic mobility of the sucrose-CaC12 nuclei slightly but decreased the mobility of the nucleoli to -2 .09 µm/s/V/cm . DNase at 50 Ag/mg protein had no effect on the mobility of the isolated sucrose-CaC12 nuclei but decreased the electrophoretic mobility of the nucleoli to -1 .21 µm/s/V/cm . RNase at 50 µg/mg protein also had no effect on the electrophoretic mobility of the sucrose-CaC12 nuclei but decreased the nucleoli mobility to -2 .10 µm/s/V/cm . Concanavalin A at 50 Ag/mg protein did not alter the nucleoli electrophoretic mobility but decreased the sucrose-CaC1 2 nuclei electrophoretic mobility to -1 .64 µm/s/V/cm . The results are interpreted to mean that the sucrose-CaC1 2 nuclear external surface contains terminal sialic acid residues in trypsin-sensitive glycoproteins, contains small amounts of hyaluronic acid, is completely devoid of nucleic acids, and binds concanavalin A . The nucleolus surface is interpreted to contain a complex made up of protein, RNA, and primarily DNA, to be devoid of sialic acid and hyaluronic acid, and not to bind concanavalin A.

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The fractionation of nuclei from mammalian cells by zonal centrifugation

Cited by 78 publications

References 11 publications

The uses of metrizamide in the fractionation of nuclei from brain and liver tissue by zonal centrifugation

The uses of metrizamide in the fractionation of nuclei from brain and liver tissue by zonal centrifugation

Effects of phthalic acid esters on the liver and thyroid.

Molecules at the External Nuclear Surface

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