Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover ( Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.     

Cortical microtubules in sweet clover columella cells developed in microgravity

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.     

Interferon-γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development

Interferon-γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF-γ addition was delayed after culture initiation. The effects of INFγ on BFU-E did not require the presence of interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1α, TNFα, or GM-CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL-1α- or TNFα-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INFγ antibody. In contrast, GM-CSF—induced suppression of BFU-E was negated by the simultaneous addition of anti-INFγ. We have previously shown that the addition of TNFα does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM-CSF—induced inhibition of BFU-E colony growth. © 1995 Wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

An increase in nitric oxide produced by rat peritoneal neutrophils is not involved in cell apoptosis

Polymorphonuclear neutrophils (PMN) obtained from carrageenin-stimulated peritoneal cavities of rats, but not blood PMN, spontaneously produced nitric oxide (NO) when incubated in vitro. Incubation of the cells with the NO synthase inhibitors, L-imino-ethyl-L-ornithine (L-NIO) or N(G)-monomethyl-L-arginine (L-NMMA), inhibited NO production. This inhibition could be reversed by L-arginine. Incubation of PMN with lipopolysaccharide (LPS) failed to enhance NO production. Pretreatment of the rats with dexamethasone (DEXA) prior to carrageenin injection or incubation of PMN with the glucocorticoid in vitro partially inhibited the spontaneous release of NO. On the other hand, when PMN obtained from DEXA pretreated rats were incubated in vitro with DEXA, NO synthase activity and hence NO generation were almost abolished. A similar inhibition was also observed following the addition of L-NIO or cycloheximide to cultures of carrageenin-elicited PMN. The NO production by PMN did not appear to be related to cell viability or apoptosis. Indeed, neither the blockade of NO generation by L-NIO nor the incubation of the neutrophils with a NO donor, S-nitroso-acetylpenicillamine (SNAP) modified the pattern of LDH release or DNA fragmentation. In summary, it appears that PMN migration triggers a continuous NO synthesis, and that NO produced by these cells is not related to their apoptosis.     

Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol.

1. Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. 2. Cell fusion was inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl, and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulphonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. 3. Gel electrophoresis of membrane proteins from "ghosts" of the erythrocytes treated with benzyl alcohol showed that a high-molecular-weight polymer was present: this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. 4. In the treated cells the proteins corresponding to bands 2 and 3 in human erythrocytes were decreased, and a polypeptide with a slightly greater mobility than band 3 was produced. 5. These changes were inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, and Tos-Lys-CH2Cl, but not by phenylmethanesulphonyl fluoride, Tos-Arg-OMe, or histamine. 6. The intramembraneous particles of the P-fracture face of cells treated with benzyl alcohol to induce fusion were decreased in number and were susceptible to cold-induced aggregation; both of these phenomena were markedly inhibited to EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. 7. These several observations indicate that a Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be essential feature of membrane fusion in this system. 8. It is suggested that this proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.