A significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility

1993 ◽  
Vol 105 (2) ◽  
pp. 433-444 ◽  
Author(s):  
C.F. Chou ◽  
C.L. Riopel ◽  
L.S. Rott ◽  
M.B. Omary
Keyword(s):  
Cell Cycle ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cytosolic Fraction ◽  
Epithelial Cell Line ◽  
Sucrose Density Gradient Centrifugation ◽  
Alternate Mechanism ◽  
10 Nm Filaments

We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

scholarly journals Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

2007 ◽  
Vol 405 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Jørgen de Jonge ◽  
Johanna M. Leenhouts ◽  
Marijke Holtrop ◽  
Pieter Schoen ◽  
Peter Scherrer ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Gradient Centrifugation ◽  
Cationic Lipid ◽  
Sucrose Density Gradient ◽  
Target Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Viral Membrane ◽  
Viral Envelopes

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

scholarly journals Isolation of 10-nm filaments from astrocytes in the mouse optic nerve.

1981 ◽  
Vol 88 (1) ◽  
pp. 245-250 ◽  
Author(s):  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa
Keyword(s):  
Optic Nerve ◽  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Sucrose Density Gradient Centrifugation ◽  
Optic Nerves ◽  
10 Nm Filaments

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.

scholarly journals Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings

1978 ◽  
Vol 169 (2) ◽  
pp. 297-303 ◽  
Author(s):  
M J Staver ◽  
K Glick ◽  
D J Baisted
Keyword(s):  
Density Gradient ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Partial Recovery ◽  
Sucrose Density Gradient Centrifugation ◽  
Freeze Thaw ◽  
Pea Seedlings ◽  
Nucleoside Diphosphatase

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain

1990 ◽  
Vol 68 (11) ◽  
pp. 1256-1261 ◽  
Author(s):  
Karen P. Farrell ◽  
Robert A. B. Keates
Keyword(s):  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Staining Intensity ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Usual Procedure ◽  
Brain Spectrin ◽  
Synapsin 1

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.Key words: microtubule-associated protein, synapsin, spectrin, tubulin, cytoskeleton.

scholarly journals Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina

1982 ◽  
Vol 203 (3) ◽  
pp. 571-575 ◽  
Author(s):  
T Tahara ◽  
Y Maeda ◽  
A Kuroiwa ◽  
K Ueno ◽  
M Obinata ◽  
...  
Keyword(s):  
Density Gradient ◽  
Storage Protein ◽  
Messenger Rna ◽  
Fat Body ◽  
Density Gradient Centrifugation ◽  
Signal Sequence ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.

scholarly journals Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1970 ◽  
Vol 119 (4) ◽  
pp. 773-784 ◽  
Author(s):  
J. A. Smith ◽  
L. Martin ◽  
R. J. B. King ◽  
M. Vértes
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Nuclear Protein ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Nuclear Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Mouse Uterus

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17β given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [14C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17β has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [14C]lysine into stromal nuclear proteins, but changes after oestradiol-17β treatment were similar to those seen in epithelium with oestradiol-17β alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17β. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17β alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-3H]oestradiol-17β by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.

scholarly journals Effect of cations on structure-linked sedimentability of lysosomal hydrolases

1968 ◽  
Vol 109 (1) ◽  
pp. 149-154 ◽  
Author(s):  
M. Anthony Verity ◽  
R. Caper ◽  
W. Jann Brown
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Lysosomal Hydrolases ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Bivalent Cation ◽  
Repeated Freezing ◽  
Triton X ◽  
Total Enzyme

1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, β-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40·7; β-glucuronidase, 51; N-acetylglucosaminidase, 39·4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na+ and K+ concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca2+ and Mg2+. 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme–membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme–membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.

Further evidence for the presence of androgen receptors in the lungs and in the head appendages of the cock

1977 ◽  
Vol 55 (3) ◽  
pp. 263-265 ◽  
Author(s):  
Jean Y. Dubé ◽  
Pierre Chapdelaine ◽  
Roland R. Tremblay
Keyword(s):  
Density Gradient ◽  
Mechanism Of Action ◽  
Androgen Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
In Vitro Binding ◽  
Vitro Binding

The presence of cytoplasmic dihydrotestosterone receptors in the lungs, the comb, the wattle, and the ear lobes of the cock was demonstrated by sucrose density-gradient centrifugation. All these tissues exhibited saturable 'in vitro' binding of dihydrotestosterone in the 8–11S region of the gradient. When 0.5 M KCl was added to the lung, wattle, and comb cytosols and to the gradients, the radioactive dihydrotestosterone migrated in the 4–5S region. These studies suggest that the mechanism of action of androgens in the head appendages of the cock and in other target tissues is similar.

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