scholarly journals Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats

1982 ◽  
Vol 204 (1) ◽  
pp. 31-36 ◽  
Author(s):  
D Sömjen ◽  
G J Sömjen ◽  
Y Weisman ◽  
I Binderman
Keyword(s):  
Density Gradient ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Macromolecular Complex ◽  
Long Bones ◽  
Newborn Rats ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation ◽  
Binding Component

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.

scholarly journals Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Jun Takezawa ◽  
Yukio Ishimi ◽  
Naomi Aiba ◽  
Kouichi Yamada
Keyword(s):  
Density Gradient ◽  
Hela Cells ◽  
Uv Light ◽  
Sucrose Density Gradient ◽  
Detectable Effect ◽  
Main Role ◽  
Template Strand ◽  
Apparent Evidence ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

Separation of paraoxon and mipafox sensitive esterases by sucrose density gradient sedimentation

1983 ◽  
Vol 17 (3-4) ◽  
pp. 315-320 ◽  
Author(s):  
Yuji Ishikawa ◽  
Edward Chow ◽  
Mark G. McNamee ◽  
Michael McChesney ◽  
Barry W. Wilson
Keyword(s):  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

ON THE MOLECULAR WEIGHT OF PREGNANT MARE'S SERUM GONADOTROPHIN

1964 ◽  
Vol 45 (4_Suppl) ◽  
pp. S163-S171 ◽  
Author(s):  
C. J. O. R. Morris
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Chromatographic Behaviour ◽  
The Cross ◽  
Active Preparation

ABSTRACT The chromatographic behaviour of pregnant mare serum and of a purified preparation of the gonadotrophin from uterine endometrial cup secretion on the cross-linked dextran gel media Sephadex G-100 and G-200 has been examined, and is consistent with a molecular weight greater than the value of 28 000 usually accepted. The sedimentation coefficient of an active preparation has been determined by sedimentation in a sucrose density gradient and serial bioassay. The value of 3.2 S for SW20 is consistent with current values. The literature data are shown to be most consistent with a molecular weight of 68 000 for the hormone, and the mechanism of its chromatography on dextran gels is discussed.

PRELIMINARY CHARACTERIZATION OF AN ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

1969 ◽  
Vol 62 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Olav Unhjem ◽  
Kjell J. Tveter ◽  
Asbjørn Aakvaag
Keyword(s):  
Proteolytic Enzymes ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Ventral Prostate ◽  
Macromolecular Complex ◽  
Sucrose Density Gradient Centrifugation

ABSTRACT Following administration of (1,2-3H)-testosterone to castrated rats or incubation of prostatic tissue with the same steroid, a gel filtration technique has been used for the isolation of a soluble steroid-macromolecular complex from the tissues. Subsequent steroid analyses revealed that 5α-androstan-17β-ol-3-one was the major component associated with the macromolecules both in the in vivo and by in vitro experiments. The complex is destroyed by proteolytic enzymes like trypsin and pronase, but is unaffected by DNase and RNase. The complex is excluded from G-200 as well as P-300 gel beds. By sucrose density gradient centrifugation two macromolecular components were found associated with radioactivity. The largest component had a sedimentation coefficient of 9.3 S and probably corresponds to the macromolecular complex demonstrated by gel filtration, whereas the smaller component had a sedimentation coefficient of 4.5 S and might represent an association of steroids with serum albumin.

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