Diffusion rates of cell surface antigens of mouse-human heterokaryons. III. Regulation of lateral diffusion rates by calcium ions

In mouse-human heterokaryons, the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membrane is enhanced by treatment of parent cells with ouabain. Ouabain treatment is ineffective if the medium lacks calcium ion, or if Verapamil, a blocker of calcium channels, is present. The divalent ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 also enhances lateral diffusion of MHC antigens, to the same extent as ouabain, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 is effective only if calcium is present in the medium. Thus it appears that increased levels of cell calcium release constraints to lateral diffusion of MHC antigens.     

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.     

Ethyl Pyruvate Inhibits HMGB1 Phosphorylation and Release by Chelating Calcium

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca²⁺ and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K⁺-induced transient [Ca²⁺]_i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca²⁺ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca²⁺ indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced intracellular Ca²⁺ surge in BV2 cells and, under this condition, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced activations of Ca²⁺-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca²⁺, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.     

Calcium control of ciliary reversal in ionophore-treated and ATP- reactivated comb plates of ctenophores

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP- reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.     

Modulation of the activity of cytosolic phospholipase A2�� (cPLA2��) by cellular sphingolipids and inhibition of cPLA2�� by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2α (cPLA2α) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2α inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2α. Phosphatidylcholine vesicles containing SM reduced cPLA2α activity. Furthermore, SM disturbed the binding of cPLA2α to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2α-dependent release of AA by inhibiting the binding of cPLA2α to glycerophospholipids.     

Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. II. Contrasting effects of local anesthetics and a calcium ionophore

In the previous study, lymphocyte surface molecules were separated into two subsets depending on whether capping was associated was associated with redistribution of cytoplasmic myosin. In the present study, the effects of the local anesthetic chlorpromazine and of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were compared. Both drugs affected the surface redistribution of immunoglobulin (Ig), Fc receptors, and the TL antigen- -molecules that appear to cap by association with microfilaments--but had no effect on the Thy.1 (theta) and H2 antigens--molecules that cap slowly, apparently unlinked to microfilament function. The capping of Ig, Fc receptor, and TL was inhibited while that of H2 and theta was not. Both drugs reversed the Ig Fc receptor, and TL caps but not the H2 and theta caps. In the former group, the reversal of caps was accompanied by a parallel reversal of the myosin segregated to the cap area. The appearance of myosin after drug treatment varied: chlorpromazine resulted in a diffuse pattern similar to that of normal lymphocytes, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced an array of aggregates and coarse filaments. The results are compatible with the view that two mechanisms for capping exist in the lymphocyte. The Ca2+ ionophore may affect capping of microfilament-dependent caps by producing a systemic activation of contractile proteins while chlorpromazine may act by disrupting a Ca2+-dependent link between surface complexes and the contractile proteins.     

Stimulation and inhibition of secretion in Paramecium: role of divalent cations

The effects of Ca2+ and Mg2+ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+-induced secretion in wild type (wt) cells was captured by "quick" fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). Quantitation of picric acid-Ca2+-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.     

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A₂α (cPLA₂α) or calcium-independent PLA₂. Surprisingly, release of arachidonic acid and LDH from cPLA₂α-deficient fibroblasts was inhibited by the cPLA₂α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca²⁺ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and H₂O₂. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.     

Epidermal growth factor-induced centrosomal separation: mechanism and relationship to mitogenesis

Using a rabbit antibody to MAP1 to stain centrosomes we have studied the mechanism by which epidermal growth factor (EGF) induces centrosomal separation in HeLa cells. The response is rapid, being detectable within 20 min after EGF (100 ng/ml) addition and by 4 h 40% of logarithmically growing cells and greater than 70% of cells synchronized at G1/S with 1 mM hydroxyurea show centrosomes separated by more than one diameter. A concentration of 0.05 ng/ml of EGF induces significant separation in synchronized cells (5-9% control vs. 20% with EGF at 0.05 ng/ml) and 0.1 to 0.5 ng/ml induces a half maximal response. Centrosomal separation is blocked by energy inhibitors, trifluoperazine, chlorpromazine, and W-7, cytochalasins B and D, and taxol, and is stimulated or enhanced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, colchicine, and oncodazole. Trifluoperazine, W-7, cytochalasin D, and taxol also block DNA synthesis in response to EGF as measured by autoradiography using [3H]thymidine. Our hypothesis based upon these results is that EGF, by raising the free calcium level, activates calmodulin, which stimulates contraction of microfilaments attached to the centrosome, pulling the daughter centrosome apart. EGF may also induce depolymerization or detachment of microtubules in the vicinity of the centrosome which ordinarily serve to maintain its position and inhibit separation. Centrosomal separation may be a key event in triggering DNA synthesis in response to EGF and colchicine.     

Characterization of the Invasive,Multidrug Resistant Non-typhoidal Salmonella Strain D23580 in a Murine Model of Infection

A distinct pathovar of Salmonella enterica serovar Typhimurium, ST313, has emerged in sub-Saharan Africa as a major cause of fatal bacteremia in young children and HIV-infected adults. {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580, a multidrug resistant clinical isolate of ST313, was previously shown to have undergone genome reduction in a manner that resembles that of the more human-restricted pathogen, Salmonella enterica serovar Typhi. It has since been shown through tissue distribution studies that {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 is able to establish an invasive infection in chickens. However, it remains unclear whether ST313 can cause lethal disease in a non-human host following a natural course of infection. Herein we report that {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 causes lethal and invasive disease in a murine model of infection following peroral challenge. The LD₅₀ of {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 in female BALB/c mice was 4.7 x 10⁵ CFU. Tissue distribution studies performed 3 and 5 days post-infection confirmed that {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 was able to more rapidly colonize the spleen, mesenteric lymph nodes and gall bladder in mice when compared to the well-characterized S. Typhimurium strain SL1344. {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 exhibited enhanced resistance to acid stress relative to SL1344, which may lend towards increased capability to survive passage through the gastrointestinal tract as well as during its intracellular lifecycle. Interestingly, {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 also displayed higher swimming motility relative to SL1344, S. Typhi strain Ty2, and the ST313 strain A130. Biochemical tests revealed that {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 shares many similar metabolic features with SL1344, with several notable differences in the Voges-Proskauer and catalase tests, as well alterations in melibiose, and inositol utilization. These results represent the first full duration infection study using an ST313 strain following the entire natural course of disease progression, and serve as a benchmark for ongoing and future studies into the pathogenesis of {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580.     

Trifluoperazine,a calmodulin antagonist,inhibits muscle cell fusion

We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5- chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1- naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion.     

Cytoplasmic pH and free Mg2+ in lymphocytes

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly- trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced no net movement of Mg2+ or H+; (b) by Mg- sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.     

Microtubules and pancreatic amylase release by mouse pancreas in vitro

The effects of vinblastine and colchicine on pancreatic acinar cells were studied by use of in vitro mouse pancreatic fragments. Vinblastine inhibited the release of amylase stimulated by bethanechol, caerulein, or ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Inhibition required preincubation with vinblastine,and maximum inhibition was observed after 90 min. Inhibition was relatively irreversible and could not be overcome by a high concentration of stimulant. Inhibition could also be produced by colchicine although longer preincubation was required and inhibition was only partial. Uptake of [3H]vinblastine and [3H]colchicine by pancreatic fragments was measured and found not to be responsible for the slow onset of inhibition by these drugs. In incubated pancreas, microtubules were present primarily in the apical pole of the cell and in association with the Golgi region. Vinblastine, under time and dose conditions that inhibited the release of stimulated amylase, also reduced the number of microtubules. The only other consistent structural effects of vinblastine were the presence of vinblastine- induced crystals and an increased incidence of autophagy. The remainder of cell structure was not affected nor were overall tissue ATP and electrolyte contents or the stimulant-induced increase in 45Ca++ efflux. It is concluded that the antisecretory effects of vinblastine and colchicine are consistent with a microtubular action, but that acinar cell microtubules are more resistant to the drugs than many other cell types.     

Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent with cancer-selective apoptogenic activity. It evokes the canonical caspase-mediated cell death pathway through death-inducing signaling complex (DISC) formation. We identified that Peroxiredoxin 6 (Prx6) interacts with caspase-10 and caspase-8 via the death effector domain (DED). Prx6 suppresses TRAIL-mediated cell death in human cancer cells, but not that induced by intrinsic apoptosis inducers such as etoposide, staurosporine, or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Among Prx1–6 members, only Prx6 binds to DED caspases and is most effective in suppressing TRAIL or DED caspase-induced cell death. The antiapoptotic activity of Prx6 against TRAIL is not likely associated with its peroxidase activity but is associated with its ability to bind to DED caspases. Increased expression of Prx6 enhances the binding of Prx6 to caspase-10 but reduces TRAIL-induced DISC formation and subsequently caspase activation. Interestingly, Prx6 is highly upregulated in metastatic gastric cancer cells, which are relatively resistant to TRAIL as compared with primary cancer cells. Downregulation of Prx6 sensitizes the metastatic cancer cells to TRAIL-induced cell death. Taken together, these results suggest that Prx6 modulates TRAIL signaling as a negative regulator of caspase-8 and caspase-10 in DISC formation of TRAIL-resistant metastatic cancer cells.     

Small molecule sensitization to TRAIL is mediated via nuclear localization,phosphorylation and inhibition of chaperone activity of Hsp27

The small chaperone protein Hsp27 confers resistance to apoptosis, and therefore is an attractive anticancer drug target. We report here a novel mechanism underlying the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizing activity of the small molecule {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, an inactive analog of the phosphoinositide 3-kinase inhibitor inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, in HeLa cells that are refractory to TRAIL-induced apoptosis. On the basis of the fact that {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 is derived from {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, itself derived from quercetin, and earlier findings indicating that quercetin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 affected Hsp27 expression, we investigated whether {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 sensitized cancer cells to TRAIL via a conserved inhibitory effect on Hsp27. We provide evidence that upon treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, Hsp27 is progressively sequestered in the nucleus, thus reducing its protective effect in the cytosol during the apoptotic process. {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511-induced nuclear translocation of Hsp27 is linked to its sustained phosphorylation via activation of p38 kinase and MAPKAP kinase 2 and the inhibition of PP2A. Furthermore, Hsp27 phosphorylation leads to the subsequent dissociation of its large oligomers and a decrease in its chaperone activity, thereby further compromising the death inhibitory activity of Hsp27. Furthermore, genetic manipulation of Hsp27 expression significantly affected the TRAIL sensitizing activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, which corroborated the Hsp27 targeting activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511. Taken together, these data indicate a novel mechanism of small molecule sensitization to TRAIL through targeting of Hsp27 functions, rather than its overall expression, leading to decreased cellular protection, which could have therapeutic implications for overcoming chemotherapy resistance in tumor cells.     

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.