c-Myc and caspase-2 are involved in activating Bax during cytotoxic drug-induced apoptosis

Activation of Bax following diverse cytotoxic stress has been shown to be an essential gateway to mitochondrial dysfunction and activation of the intrinsic apoptotic pathway characterized by cytochrome c release with caspase-9/-3 activation. Interestingly, c-Myc has been reported to promote apoptosis by destabilizing mitochondrial integrity in a Bax-dependent manner. Stress-induced activation of caspase-2 may also induce permeabilization of mitochondria with activation of the intrinsic death pathway. To test whether c-Myc and caspase-2 cooperate to activate Bax and thereby mediate intrinsic apoptosis, small interfering RNA was used to efficiently knock down the expression of c-Myc, caspase-2, and Apaf-1, an activating component in the apoptosome, in two human cancer cell lines, lung adenocarcinoma A-549 and osteosarcoma U2-OS cells. Under conditions when the expression of endogenous c-Myc, caspase-2, or Apaf-1 is reduced 80-90%, cisplatin (or etoposide)-induced apoptosis is significantly decreased. Biochemical studies reveal that the expression of c-Myc and caspase-2 is crucial for cytochrome c release from mitochondria during cytotoxic stress and that Apaf-1 is only required following cytochrome c release to activate caspases-9/-3. Although knockdown of c-Myc or caspase-2 does not affect Bax expression, caspase-2 is important for cytosolic Bax to integrate into the outer mitochondrial membrane, and c-Myc is critical for oligomerization of Bax once integrated into the membrane.     

Cofilin-mediated F-actin severing is regulated by the Rap GTPase and controls the cytoskeletal dynamics that drive lymphocyte spreading and BCR microcluster formation

When lymphocytes encounter APCs bearing cognate Ag, they spread across the surface of the APC to scan for additional Ags. This is followed by membrane contraction and the formation of Ag receptor microclusters that initiate the signaling reactions that lead to lymphocyte activation. Breakdown of the submembrane cytoskeleton is likely to be required for the cytoskeleton reorganization that drives cell spreading and for removing physical barriers that limit Ag receptor mobility. In this report, we show that Ag receptor signaling via the Rap GTPases promotes the dephosphorylation and activation of the actin-severing protein cofilin and that this results in increased severing of cellular actin filaments. Moreover, we show that this cofilin-mediated actin severing is critical for the changes in actin dynamics that drive B and T cell spreading, for the formation of BCR microclusters, and for the increased mobility of BCR microclusters within the plasma membrane after BCR engagement. Finally, using a model APC, we show that activation of this Rap-cofilin signaling module controls the amount of Ag that is gathered into BCR microclusters and that this is directly related to the magnitude of the resulting BCR signaling that is initiated during B cell-APC interactions. Thus, Rap-dependent activation of cofilin is critical for the early cytoskeletal changes and BCR reorganization that are involved in APC-dependent lymphocyte activation.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.     

A synergistic interaction of IL-6 and IL-1 mediates the thymocyte-stimulating activity produced by recombinant IL-1-stimulated fibroblasts

We characterized the ability of normal human lung fibroblasts to elaborate thymocyte-stimulating activity, spontaneously, and in response to rIL-1. Supernatants from unstimulated fibroblasts did not contain thymocyte-stimulating activity, whereas supernatants from fibroblasts incubated with rIL-1 alpha or rIL-1 beta contained more thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. This heightened thymocyte-stimulating activity was mediated by fibroblast-derived IL-6 inasmuch as it was neutralized by anti-serum against human rIL-6, and rIL-1-stimulated fibroblasts to accumulate messenger RNA for IL-6 and produce soluble IL-6 protein. However, IL-6 alone could not account for the intensity of this effect because rIL-6 only weakly stimulated thymocyte proliferation. In addition, antisera against the rIL-1 moiety that was used to prepare the supernatant had different effects on supernatants that contained and did not contain active IL-6. In the presence of IL-6 these antisera caused a greater decrease in thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. When the IL-6 was neutralized the remaining thymocyte-stimulating activity could be quantitatively accounted for and neutralized by antisera against the rIL-1 that was passively transferred. Furthermore, rIL-6 and rIL-1 (alpha or beta) synergized in stimulating thymocyte proliferation. Thus, rIL-1 stimulates fibroblasts to produce a thymocyte-stimulating activity that is largely mediated by a synergistic interaction of fibroblast-derived IL-6 and IL-1. These findings suggest that fibroblast production of IL-6 may mediate or amplify some of the tissue effects of IL-1. In addition they suggest that biologic effects previously attributed to IL-1 may be due to IL-6 alone or the concerted action of IL-1 and IL-6.     

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, -acetyldigitoxin, and -acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.Abbreviation DGT UDP-glucose:digitoxin 16-C-glucosyltransferase     

The clopidogrel after surgery for coronary artery disease (CASCADE) randomized controlled trial: clopidogrel and aspirin versus aspirin alone after coronary bypass surgery [NCT00228423]

Background

Saphenous vein graft disease remains a major limitation of coronary artery bypass graft surgery. The process of saphenous vein intimal hyperplasia begins just days after surgical revascularization, setting the stage for graft atherosclerotic disease and its sequalae. Clopidogrel improves outcomes in patients with atherosclerotic disease, and is effective at reducing intimal hyperplasia in animal models of thrombosis. Therefore, the goal of this study will be to evaluate the efficacy of clopidogrel and aspirin therapy versus aspirin alone in the prevention of saphenous vein graft intimal hyperplasia following coronary artery bypass surgery.

Methods

Patients undergoing multi-vessel coronary artery bypass grafting and in whom at least two saphenous vein grafts will be used are eligible for the study. Patients will be randomized to receive daily clopidogrel 75 mg or placebo, in addition to daily aspirin 162 mg, for a one year duration starting on the day of surgery (as soon as postoperative bleeding has been excluded). At the end of one year, all patients will undergo coronary angiography and intravascular ultrasound assessment of one saphenous vein graft as selected by randomization. The trial will be powered to test the hypothesis that clopidogrel and aspirin will reduce vein graft intimal hyperplasia by 20% compared to aspirin alone at one year following bypass surgery.

Discussion

This trial is the first prospective human study that will address the question of whether clopidogrel therapy improves outcomes and reduces saphenous vein graft intimal hyperplasia following cardiac surgery. Should the combination of clopidogrel and aspirin reduce the process of vein graft intimal hyperplasia, the results of this study will help redefine modern antiplatelet management of coronary artery bypass patients.     

Changes in gray matter induced by learning--revisited

Background

Recently, activation-dependant structural brain plasticity in humans has been demonstrated in adults after three months of training a visio-motor skill. Learning three-ball cascade juggling was associated with a transient and highly selective increase in brain gray matter in the occipito-temporal cortex comprising the motion sensitive area hMT/V5 bilaterally. However, the exact time-scale of usage-dependant structural changes occur is still unknown. A better understanding of the temporal parameters may help to elucidate to what extent this type of cortical plasticity contributes to fast adapting cortical processes that may be relevant to learning.

Principal Findings

Using a 3 Tesla scanner and monitoring whole brain structure we repeated and extended our original study in 20 healthy adult volunteers, focussing on the temporal aspects of the structural changes and investigated whether these changes are performance or exercise dependant. The data confirmed our earlier observation using a mean effects analysis and in addition showed that learning to juggle can alter gray matter in the occipito-temporal cortex as early as after 7 days of training. Neither performance nor exercise alone could explain these changes.

Conclusion

We suggest that the qualitative change (i.e. learning of a new task) is more critical for the brain to change its structure than continued training of an already-learned task.     

Effect of abscisic acid on CO2 exchange in Lemna gibba

The effects of abscisic acid (ABA) on photosynthesis, dark respiration, and photorespiration were studied in Lemna gibba L. plants. The initial concentration of ABA in the nutrient solution was 10⁻⁷M and in a few experiments, 10⁻⁶M. The cultures were grown in the same solution for time periods ranging from one hour to 12 days. Net photosynthesis, measured as CO₂ uptake by infrared gas analyser technique, was inhibited after four hours of ABA treatment and reached a minimum after four to seven days depending on the time of the year. After 12 days a substantial recovery of photosynthesis was observed. Dark respiration was significantly stimulated after two to seven days of ABA treatment but then returned to the control level. The transient effects of ABA on photosynthesis and dark respiration corresponded to the previously measured time course of [¹⁴C]-ABA uptake by Lemna . Photorespiration measured as oxygen inhibition of photosynthesis was not affected by ABA.