Triton x-100 and dmso induced cell permeability for the release of campesterol and flavonoids in cultured cells of blumea lacera (burm. F.) Dc

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
PATADE PRIYANKA ◽  
MENDHULKAR VIJAY D. ◽  
VAKIL MOINUDDIN
Keyword(s):  
Cultured Cells ◽  
Cell Permeability ◽  
Triton X ◽  
Blumea Lacera

Direct visualization of the 10-nm (100-A)-filament network in whole and enucleated cultured cells

1978 ◽  
Vol 31 (1) ◽  
pp. 393-409
Author(s):  
J.V. Small ◽  
J.E. Celis
Keyword(s):  
Cytochalasin B ◽  
Cultured Cells ◽  
Marked Effect ◽  
High Electron ◽  
Perinuclear Region ◽  
Structural Elements ◽  
3T3 Cells ◽  
10 Nm Filaments ◽  
Triton X ◽  
Filament Network

Following extraction of actomyosin and tubulin from cultured cells treated with Triton X-100, a cytoskeleton remains which is composed predominantly of the cell nucleus encompassed by a network of 10-nm filaments. After negative staining the dense perinuclear region appears as a densely woven filament net punctuated by patches of high electron density. Enucleation of 3T3 cells with cytochalasin B gives rise to karyoplasts surronunded by 10-nm filaments and cytoplasts in which 10-nm filaments remain situated in the central region of the cytoplasm. While the 10-nm filaments occurred mainly as single filaments in human skin fibroblasts and 3T3 cells, in epithelioid PtK1 and PtK2 cells they were commonly associated in prominent meandering bundles. In addition, in these latter cells after Triton extraction the remaining ribosomes were bound specifically to the 10-nm-filament net. After exposure of 3T3 cells to cytochalasin B the 10-nm filaments formed branches that radiated from the perinuclear region into the immobile cell extensions. Concavalin A had no marked effect on the distribution of the 10-nm-filament net. The results suggest that the 10-nm filaments act primarily as structural elements, serving, in particular, to support and constrain the nucleus in its position in the cell.

Plasminogen Activator (PA) Activity In Metastatic Cells: An Amidolytic Approach

1981 ◽  
Author(s):  
D Coen ◽  
A Bini ◽  
G Balconi ◽  
F Delaini ◽  
L Mussoni ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Fibrinolytic Activity ◽  
Tumor Tissue ◽  
Cultured Cells ◽  
Cell Extracts ◽  
Mechanical Disruption ◽  
Metastatic Cells ◽  
Triton X ◽  
Selection Of

It has been proposed that fibrinolytic activity can play an important role in the process of metastasis formation. Nevertheless, it is not yet clear in which phase of the tumor growth and dissemination this activity is involved. We measured the fibrinolytic activity of cells from primary tumor and metastatic nodules of 3LL, an i.m. implanted murine tumor which selectively metastasizes to the lungs. Tumor cells have been studied both immediately after mechanical disruption of tumor tissue and after in vitro culturing to confluence. Their P.A. activity was tested by an amidolytic assay in which cells were incubated with purified plasminogen (3CU/ml) and 4mM S-2251 (Kabi Diagnostica, Stockholm, Sweden), a plasmin specific chromogenic substrate. After 3 hour incubation at 37°C, the reaction was stopped with acetic acid and absorbance read at 405 nm.Cells from the primary tumor and metastatic nodules showed a similar fibrinolytic activity, which was in both cases in- increased 3 to 4 fold in cell extracts obtained after preincubation with TRITON X-100. A dose-response curve plotted with increasing urokinase concentrations showed a parallel course. This data suggests that, in the 3LL model, PA activity is not one of the properties characterizing the selection of metastatic cells.On the other hand,cultured cells presented consistently higher levels of PA than their native counterparts, suggesting that adhesion of cells in culture may stimulate PA production or, alternatively, that cultured cells are a selected population in comparison to the overall number of native cells.

Cadmium inhibits glucose uptake in primary cultures of mouse cortical tubule cells

1990 ◽  
Vol 258 (6) ◽  
pp. F1625-F1633 ◽  
Author(s):  
S. S. Blumenthal ◽  
D. L. Lewand ◽  
M. A. Buday ◽  
J. G. Kleinman ◽  
S. K. Krezoski ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glucose Uptake ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Defined Medium ◽  
Isobutyric Acid ◽  
Cell Permeability ◽  
Dependent Manner ◽  
Lactate Dehydrogenase Activity ◽  
Tubule Cells

We studied the effect of cadmium (Cd2+) on transport of alpha-methylglucoside in primary cultures of mouse kidney cortical tubule cells grown in defined medium. When cultured cells were exposed to Cd2+ concentrations from 0 to 6 microM for 24 h, uptake of alpha-methylglucoside was inhibited in a dose-dependent manner by up to 50%. By contrast, acute exposure of the cells to 7 microM Cd2+ for 60 min did not inhibit alpha-methylglucoside uptake. Increasing Cd2+ concentrations progressively decreased the Vmax of Na(+)-dependent glucose cotransport but not the Km for glucose. Cell ATP/ADP ratios of unexposed monolayers and of cells exposed to 4.5 microM Cd2+ for 24 h were 5.0 and 4.9, respectively (n = 3). Intracellular volume, lactate dehydrogenase activity, and cell Na+ and K+ concentrations were unaltered even after 24 h of exposure to 7 microM Cd2+. Untreated and Cd2+-treated monolayers preloaded with alpha-methylglucoside released the sugar analogue into the medium at nearly identical rates, indicating that Cd2+ did not alter cell permeability to glucose. Uptake of the amino acid analogue alpha-(methylamino)isobutyric acid was not affected by prior Cd2+ exposure. Whereas cell DNA content declined in Cd2(+)-exposed plates, both Na(+)-glucose and Na(+)-amino acid cotransport were enhanced at lower cell densities. Protein and DNA synthesis, estimated, respectively, by incorporation of [3H]leucine and [3H]thymidine into acid-insoluble material, were not significantly affected at 6 microM Cd2+. We conclude that after a lag time Cd2+ selectively inhibits renal Na(+)-dependent glucose transport despite an unchanged gradient for Na+ across the cell membrane.

Association of calmodulin with lysosomes

1987 ◽  
Vol 87 (2) ◽  
pp. 327-336
Author(s):  
T.B. Nielsen ◽  
J.B. Field ◽  
J.R. Dedman
Keyword(s):  
Cultured Cells ◽  
Membrane Vesicle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Resting Cells ◽  
Follicular Cells ◽  
Cytoplasmic Staining ◽  
Thyroid Follicular Cells ◽  
Triton X ◽  
Spindle Fibres ◽  
Mitotic Cells

We examined the subcellular localization of calmodulin in several cultured cells (primary thyroid follicular cells, thyroid C-cell tumour cells (TT), kidney cells (PtK2-L23) and peritoneal macrophages) by indirect immunofluorescence using affinity-purified antibody to calmodulin. When cells were fixed with 3% formaldehyde for 15 min prior to lysis with 0.5% Triton X-100, spindle fibres in mitotic cells were fluorescent and a diffuse cytoplasmic localization of calmodulin was observed in resting cells. However, when cells were lysed with 0.5% Triton X-100 for 90s prior to fixation for 30 min with 3% formaldehyde, three effects were observed. One: there was little diffuse cytoplasmic staining. Two: discrete vesicles were stained. Three: spindle fibres in mitotic cells were fluorescent. The stained vesicles were phase-dense and ranged from 0.1 to 0.5 micron in primary thyroid follicular cells but were smaller in PtK2 and TT cells. The thyroid follicular cells retained vesicular staining after exposure to thyrotropin and isobutylmethylxanthine, but the number of labelled vesicles decreased by almost 80%. Phase-dense vesicles were identified as lysosomes or other acidic vesicles by vital staining with Acridine Orange. After differential centrifugation of thyroid homogenates, calmodulin was measured by radioimmunoassay (RIA) and found in both cytosolic (89%) and membrane vesicle fractions (11%). The vesicular calmodulin was not eluted by washing with 5 mM-EGTA. The thyroid fractions were subjected to SDS-polyacrylamide gel electrophoresis and the gels incubated with 125I-labelled calmodulin to reveal calmodulin acceptor proteins (CAPs). The vesicle fraction contained quantitatively major CAPs with Mr of 200,000, 140,000, 89,000, 38,000 and 34,000, and minor CAPs of 60,000 and 50,000. Washing the pellet with 5 mM-EGTA did not reduce the content of CAPs. Thus, calmodulin and CAPs are present both in the cytoplasm and in a membrane vesicle fraction. The lysosomal locale of calmodulin and the effect of thyrotropin on vesicle number suggest a role for calcium in the regulation of lysosome function.

scholarly journals Purification of Lipid Rafts from Cultured Cells

2002 ◽  
Vol 2 ◽  
pp. 1662-1666 ◽  
Author(s):  
John Graham
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Microsomal Fraction ◽  
Cultured Cells ◽  
The Other ◽  
Low Concentrations ◽  
Nonionic Detergent ◽  
Triton X ◽  
Intact Lipid

Lipid-rich lipid rafts are microdomains of the plasma membrane that are resistant to low concentrations of nonionic detergent. This forms the basis for their isolation. Either a microsomal fraction or a postnuclear supernatant are loaded beneath a discontinuous iodixanol gradient. If all the solutions contain 0.5–1.0% Triton X-100, the intact lipid rafts float to the top of the gradient while all of the other detergent-solubilized membranes remain at the bottom.

Deep etching of rapidly frozen cytoskeletons of motile fibroblasts

1987 ◽  
Vol 45 ◽  
pp. 900-901
Author(s):  
Julian P. Heath ◽  
Frédérique Bard
Keyword(s):  
Cultured Cells ◽  
Three Dimensional ◽  
Rapid Freezing ◽  
Deep Etching ◽  
Dimensional Network ◽  
Detergent Extraction ◽  
Chick Embryo Heart ◽  
Embryo Heart ◽  
Triton X ◽  
Whole Mounts

The cytoskeleton of motile cultured cells is a complex three dimensional network of actin microfilaments, microtubules and intermediate filaments. We are using rapid freezing and deep etching to visualise this network in human and chick fibroblasts after detergent extraction. This method offers high resolution, high contrast images that retain three dimensionality with minimal artefact, and gives views of the cytoskeleton that differ from those obtained with more conventional techniques such as HVEM of thick plastic sections, or CTEM of critical point dried whole mounts.Human skin fibroblasts and chick embryo heart fibroblasts were grown on No. 1 glass coverslips and fixed and extracted with 0.1 M PIPES buffer pH 7 containing 0.25% glutaraldehyde, 0.5% Triton X-100, 1 mM MgCl2, 1 mM EGTA, 1 μg/ml phalloidin and 5 μg/ml taxol, for 30 s to 10 min at RT; further fixed in 2.5% glutaraldehyde in PIPES for 10 min, postfixed in 1% osmium in PIPES for 5 min and dehydrated in 70% ethanol.

scholarly journals Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells.

1989 ◽  
Vol 108 (4) ◽  
pp. 1453-1463 ◽  
Author(s):  
K K Pfister ◽  
M C Wagner ◽  
D L Stenoien ◽  
S T Brady ◽  
G S Bloom
Keyword(s):  
Monoclonal Antibodies ◽  
Primary Cilia ◽  
Cultured Cells ◽  
Cell Types ◽  
Bovine Brain ◽  
Double Labeling ◽  
Organelle Motility ◽  
Transient Interactions ◽  
Cytoplasmic Structures ◽  
Triton X

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.

Co-determination of ATP and proteins in Triton X 100 non-ionic detergent-opened monolayer cultured cells

Luminescence ◽  
2007 ◽  
Vol 22 (5) ◽  
pp. 415-419 ◽  
Author(s):  
Tamás Kőszegi ◽  
József Petrik ◽  
Sanda Vladimir-Knežević ◽  
Sándor Nagy
Keyword(s):  
Cultured Cells ◽  
Ionic Detergent ◽  
Triton X

Golgi membrane skeleton: identification, localization and oligomerization of a 195 kDa ankyrin isoform associated with the Golgi complex

1997 ◽  
Vol 110 (10) ◽  
pp. 1239-1249 ◽  
Author(s):  
K.A. Beck ◽  
J.A. Buchanan ◽  
W.J. Nelson
Keyword(s):  
Golgi Complex ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Affinity Purification ◽  
Light And Electron Microscopy ◽  
Brefeldin A ◽  
Membrane Skeleton ◽  
Coat Proteins ◽  
Golgi Membrane ◽  
Triton X

To extend our finding of a Golgi-localized form of the membrane skeleton protein spectrin, we have identified an isoform of ankyrin that associates at steady state with the Golgi complex. Immuno-light and -electron microscopy show that this ankyrin isoform localizes to the perinuclear cytoplasm on tubular vesicular structures that co-stain with Golgi marker proteins. An antiserum raised against erythrocyte ankyrin, which was used to identify the Golgi ankyrin, recognized three prominent polypeptides of 220, 213 and 195 kDa in MDCK cells. Affinity purification of this antiserum against each of these MDCK cell ankyrins revealed that only an antibody specific for the 195 kDa form retained the ability to stain the Golgi complex; affinity purified antibody preparations specific for both the 220 and 213 kDa forms stained punctate and reticular cytoplasmic structures distinct from the Golgi complex. Antibody specific for the 195 kDa ankyrin did not recognize a recently identified 119 kDa ankyrin that is also localized to the Golgi. The 195 kDa Golgi ankyrin binds purified erythrocyte spectrin, and rapidly co-sediments with Golgi beta-spectrin during brief, low speed centrifugation of Triton X-100 extracts of MDCK cells. Golgi ankyrin and beta-spectrin are retained on tubular vesicular ‘Golgi ghosts’ following extraction of cultured cells with Triton X-100. Significantly, Golgi ghost tubules containing ankyrin/spectrin are co-linear with individual microtubules, suggesting a role for both Golgi membrane skeleton and microtubules in spatial localization of the Golgi. Golgi ankyrin dissociates from Golgi membranes during mitosis and in cells treated with brefeldin A, indicating that Golgi ankyrin has a dynamic assembly state similar to that of Golgi spectrin and other Golgi membrane coat proteins.

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