Action of Cytochalasin B on Cultured Human Lymphocytes

Smith, George F.; Ridler, M. A. C.; Faunch, Janet A.

doi:10.1038/2161134a0

Cited by 32 publications

(17 citation statements)

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“…5). As in other tissues (2,4) (19,20) and lymphocytes (21,22) by blocking cytoplasmic cleavage after nuclear division. The large, multinucleated lymphocytes cultured (6 days) -in the presence of cytochalasin B (see Methods) can specifically bind about 2.4 X 10-15 mol of insulin per cell, or about 10 times more than the control, transformed lymphocytes.…”

Section: Methodsmentioning

confidence: 99%

Emergence of Insulin Receptors on Human Lymphocytes During In Vitro Transformation

Krug

Cuatrecasas

1972

Proc. Natl. Acad. Sci. U.S.A.

140

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Essentially no specific binding sites for insulin are detected in small lymphocytes freshly isolated from human blood. Insulin-binding sites appear on the lymphocyte surface during transformation in vitro with concanavalin A, and the number of these receptors increases sharply to reach a maximum between 24 and 46 hr after exposure to the mitogen. The The binding of insulin to its receptor sites on fat cells and on liver and fat-cell membranes has been studied extensively (1-8). Insulin-receptor sites on cells other than the typical target cells for insulin, namely human circulating cells and cultured fibroblasts, have recently been described (9). The present studies demonstrate that while no significant specific binding of insulin is detectable on normal peripheral blood lymphocytes of humans, a dramatic appearance of insulin receptors occurs during lymphocyte transformation induced by the plant mitogen concanavalin A. The short-term lymphocyte culture provides a useful model for investigation of changes in the number and properties of surface receptor sites of cells as they undergo functional changes such as metabolic activation, differentiation, and cell division. The present studies are also of interest in view of the profound insulin-like biological properties of concanavalin A (10), the inhibition by insulin (11, *) and by concanavalin A (10, *) of * Tell, G. & Cuatrecases, P., submitted to J. Biol. Chem. 2604 adenylate cyclase activity in isolated membrane preparations, the growth-promoting properties of insulin in various mammalian cells in tissue culture (12,15), and the important relationships that apparently exist between cyclic AMP and cell growth (15-18). METHODSLymphocytes were separated from other leukocytes by passage of freshly drawn, heparinized blood (200-500 ml) from a healthy donor through a sterile, disposable nylon-fiber column (Fenwal Laboratories) that was immediately prewashed (at 370) with 250 ml of sterile normal saline. The erythrocytes (in 10-ml aliquots) were allowed to settle by gravity at 370 and the lymphocyte-rich plasma was collected before the appearance of a visible buffy coat. For removal of excess platelets, the plasma was centrifuged at 150 X g for 15 min, and the pellet was washed 2-3 times with Medium 199 containing 5% fetal-bovine serum. The lymphocytes, recovered in 60% yield, constituted 95-98% of the cells; 1-5% were granulocytes and less than 1% were monocytes. Platelets did not exceed 50, and erythrocytes did not exceed 200 per 100 leukocytes. Leukocyte counts (modified Neubauer hemocytometer) were based on a count of at least 700 cells. For differential counts (500 cells, three slides), samples on pulled coverslips were fixed in 99% methanol and stained with Giemsa stain. Increased cell size, characteristic nuclear staining, multiple nucleoli, and cytoplasmic basophilia were used to judge blastic transformation.For culture the cells were suspended in Medium 199 supplemented with 8% fresh homologous serum containing 100 units of penicillin and 50 jig of s...

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Section: Methodsmentioning

confidence: 99%

Emergence of Insulin Receptors on Human Lymphocytes During In Vitro Transformation

Krug

Cuatrecasas

1972

Proc. Natl. Acad. Sci. U.S.A.

140

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“…[8] In the current study,t he fermentation broth of Aspergillus flavipes was phytochemically investigated, leading to the isolation and characterization of four novel cytochalasan alkaloids (asperchalasines A-D, 1-4; Figure 1). Asperchalasine A( 1), which is the first example of ac ytochalasan dimer generated by the fusion of two cytochalasan molecules to an epicoccine,possesses an unprecedented 13-oxatetracyclo[7.2.1.1 2,5 .0 1,6 ]tridec-8,12-dione core containing as many as 20 chiral centers.S tructurally,t he dimeric and polymeric features of 1 are sufficient to make it stand out not only from the large family of cytochalasans,but also from the naturally occurring alkaloids.M eanwhile, asperchalasines B-D (2-4)s eem to be the biosynthetic intermediates of 1 and are ap robable biogenetic pathway for 1 from monomeric cytochalasan.…”

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confidence: 99%

“…Abstract: Asperchalasine A( 1), the first cytochalasan dimer featuring aunique decacyclic 5/6/11/5/5/6/5/11/6/5 ring system consisting of 20 chiral centers,w as isolated from the culture broth of Aspergillus flavipes.T hree biogenetically related intermediates,a sperchalasines B-D (2)(3)(4), were also isolated. Their structures,i ncluding their absolute configurations,w ere elucidated using ac ombination of HRESIMS,N MR, ECD, molecular modeling,a nd single-crystal X-rayd iffraction techniques.C ompound 1,w hichp ossesses an unprecedented 13-oxatetracyclo[7.2.1.1 2,5 .0 1,6 ]tridec-8,12-dione core structure, is the first example of ad imeric cytochalasan alkaloid. The biogenetic pathways of 1-4 were described starting from the co-isolated compounds 5 and 6.M ore importantly, 1 induced significant G1-phase cell cycle arrest by selectively inhibiting cyclin A, CDK2 and CDK6 in cancerous,but not normal, cells, highlighting it as ap otentially selective cell cycle regulator against cancer cells.…”

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Asperchalasine A, a Cytochalasan Dimer with an Unprecedented Decacyclic Ring System, from Aspergillus flavipes

et al. 2015

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Asperchalasine A (1), the first cytochalasan dimer featuring a unique decacyclic 5/6/11/5/5/6/5/11/6/5 ring system consisting of 20 chiral centers, was isolated from the culture broth of Aspergillus flavipes. Three biogenetically related intermediates, asperchalasines B-D (2-4), were also isolated. Their structures, including their absolute configurations, were elucidated using a combination of HRESIMS, NMR, ECD, molecular modeling, and single-crystal X-ray diffraction techniques. Compound 1, which possesses an unprecedented 13-oxatetracyclo[7.2.1.1(2,5).0(1,6)]tridec-8,12-dione core structure, is the first example of a dimeric cytochalasan alkaloid. The biogenetic pathways of 1-4 were described starting from the co-isolated compounds 5 and 6. More importantly, 1 induced significant G1-phase cell cycle arrest by selectively inhibiting cyclin A, CDK2 and CDK6 in cancerous, but not normal, cells, highlighting it as a potentially selective cell cycle regulator against cancer cells.

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“…Finally, many effects of cytochalasin B are difficult to explain solely in terms of an effect on filament systems. (i) Nuclei are shunted out of cells on long cytoplasmic stalks and are sometimes even extruded from these cells (1,24); (ii) mitotic rate is reduced (1,6); (iii) nuclei are displaced toward the periphery of myotubes (6); and (iv) the adhesion of blood platelets is altered (21). Thus, since F-actin is unaffected structurally and functionally, since the HMM binding and release are unaffected, and since there is no evidence that cytochalasin B affects the myosin component in cellular cytoplasm, we must conclude that we do not know yet the sites of action of cytochalasin B.…”

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Cytochalasin B: Does It Affect Actin-Like Filaments?

Forer¹,

Emmersem²,

Behnke³

1972

Science

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An in vitro system was used to test the purported action of cytochalasin B. At concentrations 100 times those used for experiments in vivo, cytochalasin B did not cause the breakdown of F-actin, did not inhibit the transformation of G-actin to F-actin, did not inhibit the binding of heavy meromyosin to F-actin, and did not inhibit the adenosine triphosphate-induced release of heavy meromyosin from F-actin.

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Action of Cytochalasin B on Cultured Human Lymphocytes

Cited by 32 publications

References 3 publications

Emergence of Insulin Receptors on Human Lymphocytes During In Vitro Transformation

Emergence of Insulin Receptors on Human Lymphocytes During In Vitro Transformation

Asperchalasine A, a Cytochalasan Dimer with an Unprecedented Decacyclic Ring System, from Aspergillus flavipes

Cytochalasin B: Does It Affect Actin-Like Filaments?

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