Maintaining Cell Membrane Integrity Research Articles

Hypobaric storage reduced core browning of Yali pear fruits

Core browning is a major issue of pear fruits. The primary factor leading to development of core browning is high levels of CO2 in storage. Hypobaric storage is a new postharvest technique to extend the shelf life of fruits and can reduce CO2 partial pressure. In this study, the effect of hypobaric storage on core browning of Yali pear fruits (Pyrus bretschneideri Rehd.) was evaluated. Results showed that fruits after 0.025MPa hypobaric treatment had 37.3% lower rate of core browning development and higher acidity and soluble solid content than control fruits. In addition, 0.025MPa hypobaric treatment increased activities of cytochrome oxidase (CCO) and peroxidase (POD), and 2,2-diphenylpicrylhydrazyl (DPPH) radicals and superoxide anion scavenging capabilities. In comparison with the control, hypobaric treated fruits had lower activity of polyphenol oxidase (PPO) and higher level of total polyphenols. Hypobaric treatment also reduced an increase of succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities. Results indicated that hypobaric treatment could control core browning of pear fruits by regulating reactive oxygen species (ROS) generation and elimination enzymes to maintain cell membrane integrity and reduce enzymatic browning.

Effects of nutrient and water restriction on thermal tolerance: A test of mechanisms and hypotheses

Nutritional deprivation or desiccation can influence thermal tolerance by impacting the insects' ability to evaporatively cool, maintain cell membrane integrity and conduct protective or repair processes. Recovery from chilling is also linked to the re-establishment of iono- and osmo-regulatory homeostasis. Here, using Mediterranean fruit fly (Ceratitis capitata, Diptera: Tephritidae), we manipulated water and nutrient availability to test the mechanistic expectation that changes in whole organism lipid and water content can elicit variation in cold or heat tolerance (scored as chill coma recovery time and heat knockdown time). We measured body condition (body water and lipid content) as well as heat shock protein 70 gene (hsp70) and protein (HSP70) levels. A significant reduction in body water content with water restriction did not translate into differences in chill coma recovery. When nutrient restriction was coupled with water deprivation, this resulted in a significant reduction (−54%) of heat knockdown time in females but male flies were unaffected. There was no evidence for an hsp70 or HSP70 response under any of the stress treatments and therefore no correlation with heat or cold tolerance. Heat hardening decreased all hsp levels. Therefore, although body water and total body lipid content differed between the treatment groups, the contribution of these factors to thermal tolerance was inconsistent with mechanistic expectations in heat knockdown time and insignificant for chill coma recovery. These results therefore highlight that the effects of resource restriction on thermal limits in insects are mechanistically more complex than previous models of stress resistance have suggested.

Pre-storage cold acclimation maintained quality of cold-stored cucumber through differentially and orderly activating ROS scavengers

Cucumber fruit are susceptible to chilling injury (CI) during storage and shipping at temperatures below 7–10°C. Pre-storage cold acclimation (PsCA) is a promising postharvest method for reducing CI and maintaining quality, but the reported duration of PsCA varies greatly, partly because no method so far has been developed for evaluating the chilling tolerance induced by PsCA at an early time. Following PsCA at 10°C for 6h, 12h, 24h, 48h and 72h, CI, secondary diseases and relative electrolyte leakage of cucumbers stored at 5°C decreased with PsCA duration, indicating PsCA-induced chilling tolerance in cucumber was positively correlated with the length of exposure to PsCA. Meanwhile, PsCA increased soluble solids and ascorbic acid, and reduced malondialdehyde, O2− and H2O2, suggesting that PsCA improved quality by inhibiting reactive oxygen species (ROS) and maintaining cell membrane integrity. In addition, PsCA enhanced gene expression and activities of four enzymatic ROS scavengers, superoxide dismutase, peroxidase, ascorbate peroxidase, catalase and two non-enzymatic ROS scavengers, AsA and glutathione. However, different ROS scavengers had variable transition points before they were consistently activated, suggesting that ROS scavengers were differentially activated in an orderly way and that the chilling tolerance is positively correlated with the number of activated ROS scavengers, but require enough number of ROS scavengers to be effective. This study provides possibility for early evaluation of PsCA-induced chilling tolerance and for timely arrangement of marketing and processing to reduce losses.

Sugars as supplemental cryoprotectants for marine organisms

AbstractCryopreservation has been extensively used in various mega‐industries and has recently been applied in genetic banking for conservation purposes. Compared with conventional cell preservation methods, cryopreservation can maintain the viability of cryopreserved cells for an indefinite time at a relatively lower cost and lesser manpower, with lower probabilities of contamination and genetic changes. This study presents the crucial role of sugar, a cryoprotectant supplement in cryopreservation. Sugar molecules typically interact with the lipid bilayer during the freezing phase to maintain plasma membrane integrity when cells undergo dehydration. When combined with other permeable cryoprotectants such as glucose with methanol and trehalose with dimethyl sulphoxide, sugar prolongs and enhances cellular post‐thaw viability. Moreover, terrestrial and marine organisms have benefited from the inclusion of sugar in the cryopreservation protocol. For wide range of cells such as the sperm, oocytes, embryos and larvae of marine vertebrates and invertebrates, as well as marine algae, cryopreservation with sugar produced positive results compared with cryopreservation without sugar. Not all sugar is beneficial, and the type and concentration of sugar should be applied according to the species. Moreover, the freezing method may also affect the function of sugar. Nevertheless, understanding the role of sugar in cryobiology and conducting a preliminary trial of sugar for cryopreserving cells would benefit future research on cryopreservation.

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism.

How are necrotic cells recognized by their predators?

Necrosis is a type of cell death often caused by cell injury and is linked to human diseases including neuron degeneration, stroke, and cancer. Cells undergoing necrosis are engulfed and degraded by engulfing cells, their predators. The mechanisms by which necrotic cells are recognized and removed remain elusive. Here we comment on our recent findings that reveal new molecular mechanisms of necrotic-cell recognition. Through studying the C. elegans touch neurons undergoing excitotoxic necrosis, we identified a receptor/ligand pair that enables engulfing cells to recognize necrotic neurons. The phagocytic receptor CED-1 is activated through interaction with its ligand phosphatidylserine (PS), exposed on the surface of necrotic cells. Furthermore, against the common belief that necrotic cells have ruptured plasma membrane, we found that necrotic C. elegans touch neurons actively present PS on their outer surfaces while maintaining plasma membrane integrity. We further identified 2 mechanisms governing the presentation of PS, one of which is shared with cells undergoing apoptosis, a “cell suicide” event, whereas the other is unique to necrotic neurons. The influx of Ca2+, a key necrosis-triggering factor, is implicated in activating a neuronal PS-scramblase for PS exposure. We propose that the mechanisms controlling PS-exposure and necrotic-cell recognition by engulfing cells are likely conserved from worms to humans.

Characterisation of Lipid Changes in Ethylene-Promoted Senescence and Its Retardation by Suppression of Phospholipase Dδ in Arabidopsis Leaves

Ethylene and abscisic acid (ABA) both accelerate senescence of detached Arabidopsis leaves. We previously showed that suppression of Phospholipase Dδ (PLDδ) retarded ABA-promoted senescence. Here, we report that ethylene-promoted senescence is retarded in detached leaves lacking PLDδ. We further used lipidomics to comparatively profile the molecular species of membrane lipids between wild-type and PLDδ-knockout (PLDδ-KO) Arabidopsis during ethylene-promoted senescence. Lipid profiling revealed that ethylene caused a decrease in all lipids levels, except phosphatidic acid (PA), caused increases in the ratios of digalactosyl diglyceride/monogalactosyl diglyceride (MGDG) and phosphatidylcholine (PC)/phosphatidylethanolamine (PE), and caused degradation of plastidic lipids before that of extraplastidic lipids in wild-type plants. The accelerated degradation of plastidic lipids during ethylene-promoted senescence in wild-type plants was attenuated in PLDδ-KO plants. No obvious differences in substrate and product of PLDδ-catalyzed phospholipid hydrolysis were detected between wild-type and PLDδ-KO plants, which indicated that the retardation of ethylene-promoted senescence by suppressing PLDδ might not be related to the role of PLDδ in catalyzing phospholipid degradation. In contrast, higher plastidic lipid content, especially of MGDG, in PLDδ-KO plants was crucial for maintaining photosynthetic activity. The lower relative content of PA and higher PC/PE ratio in PLDδ-KO plants might contribute to maintaining cell membrane integrity. The integrity of the cell membrane in PLDδ-KO plants facilitated maintenance of the membrane function and of the proteins associated with the membrane. Taking these findings together, higher plastidic lipid content and the integrity of the cell membrane in PLDδ-KO plants might contribute to the retardation of ethylene-promoted senescence by the suppression of PLDδ.

Influence of gum arabic coating enriched with calcium chloride on physiological, biochemical and quality responses of mango (Mangifera indica L.) fruit stored under low temperature stress

Effects of gum arabic (GA) 10% and calcium chloride (CA) 3% on the physiological, biochemical and quality responses of mango (Mangifera indica L. cv. Choke Anan) fruit were investigated. Fruit were stored at 6°C and 90% relative humidity for 28 d and then transferred to 25°C for an additional 5 d shelf life. Significant (P≤0.05) differences were observed in fruit treated with CA 3% and GA 10% as compared to the control. The combined treatment of CA 3% and GA 10%, significantly alleviated chilling injury, malondialdehyde (MDA) content and electrolyte leakage than the control fruit. This treatment reduced the increase in hydrogen peroxide (H2O2) content, superoxide anion (O2−) production rate and enhanced DPPH radical scavenging activity. Furthermore, GA 10% alone or in combination with CA 3% effectively inhibited the loss of total phenolic content and ascorbic acid. The result of transmission electron microscopy confirmed that treated fruit maintained cell membrane integrity. These results suggest that application of GA 10% coating combined with CA 3% might be enhanced low temperature tolerance by improving the antioxidant defense system and reducing oxidative damage of mango fruit.

Spontaneous Packaging and Hypothermic Storage of Mammalian Cells with a Cell-Membrane-Mimetic Polymer Hydrogel in a Microchip.

Currently, continuous culture/passage and cryopreservation are two major, well-established methods to provide cultivated mammalian cells for experiments in laboratories. Due to the lack of flexibility, however, both laboratory-oriented methods are unable to meet the need for rapidly growing cell-based applications, which require cell supply in a variety of occasions outside of laboratories. Herein, we report spontaneous packaging and hypothermic storage of mammalian cells under refrigerated (4 °C) and ambient conditions (25 °C) using a cell-membrane-mimetic methacryloyloxyethyl phosphorylcholine (MPC) polymer hydrogel incorporated within a glass microchip. Its capability for hypothermic storage of cells was comparatively evaluated over 16 days. The results reveal that the cytocompatible MPC polymer hydrogel, in combination with the microchip structure, enabled hypothermic storage of cells with quite high viability, high intracellular esterase activity, maintained cell membrane integrity, and small morphological change for more than 1 week at 4 °C and at least 4 days at 25 °C. Furthermore, the stored cells could be released from the hydrogel and exhibited the ability to adhere to a surface and achieve confluence under standard cell culture conditions. Both hypothermic storage conditions are ordinary flexible conditions which can be easily established in places outside of laboratories. Therefore, cell packaging and storage using the hydrogel incorporated within the microchip would be a promising miniature and portable solution for flexible supply and delivery of small amounts of cells from bench to bedside.

Emerging roles of apoptotic microtubules during the execution phase of apoptosis.

Apoptosis is a genetically programmed energy-dependent process of cell demise, characterized by specific morphological and biochemical events in which the activation of caspases has an essential role. During apoptosis the cytoskeleton participates actively in characteristic morphological rearrangements of the dying cell. This reorganisation has been assigned mainly to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent reports have showed that microtubules are reformed during the execution phase of apoptosis organizing an apoptotic microtubule network (AMN). AMN is organized behind plasma membrane, forming a cortical structure. Apoptotic microtubules repolymerization takes place in many cell types and under different apoptotic inducers. It has been hypothesized that AMN is critical for maintaining plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disorganization leads apoptotic cells to secondary necrosis and the release of potential toxic molecules which can damage neighbor cells and promotes inflammation. Therefore, AMN formation during physiological apoptosis or in pathological apoptosis induced by anti-cancer treatments is essential for tissue homeostasis and the prevention of additional cell damage and inflammation.

The RNA in reticulocytes is not just debris: It is necessary for the final stages of erythrocyte formation

Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

Methyl jasmonate induced defense responses increase resistance to Fusarium oxysporum f. sp. cubense race 4 in banana

Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum, is a severe banana (Musa spp.) root disease, currently threatening banana production worldwide. Methyl jasmonate (MeJA) has been recognized for its ability to induce systemic defense against various pathogens. In this study, we treated banana plantlets with 1.5mM exogenous MeJA. The effects of treatment on the level of chitinase, β-1,3-glucanase, total phenols (TP), hydrogen peroxide (H2O2), superoxide radicals (O2−), malondialdehyde (MDA), as well as the activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT) and phenylalanine ammonia-lyase (PAL), plus disease incidence (DI) and disease severity (DS) were investigated. The results demonstrated that MeJA regulated the contents of H2O2 and O2−, and decreased MDA in banana roots. Meanwhile, plantlets treated with MeJA exhibited considerably higher chitinase, β-1,3-glucanase and TP contents, and higher activities of SOD, POD, PPO, CAT and PAL. In addition, MeJA treatment dramatically reduced disease incidence (DI) and disease severity (DS) of banana plants compared to control. The higher activities of scavenger antioxidant enzymes account for the reduction of cellular levels of reactive oxygen species (ROS), which is of vital importance for maintaining cell membrane integrity. The accumulation of pathogenesis-related proteins, chitinases and β-1,3-glucanase, as well as total phenols and PAL activity in the treated roots also indicated that MeJA triggered key enzymes of secondary metabolite biosynthetic pathways, suggesting that MeJA was involved in the activation of a disease-related defense system.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na+/K+ pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.

High Throughput Single-cell and Multiple-cell Micro-encapsulation

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Postharvest BTH treatment induced disease resistance and enhanced reactive oxygen species metabolism in muskmelon (Cucumis melo L.) fruit

Benzo (1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a novel synthetic elicitor capable of inducing disease resistance in many plants. Fruits were dipped in BTH at 100 mg/L for 10 min, and then stored at room temperature (22 ± 2 °C, relative humidity 55–60 %). The results showed that BTH significantly (P < 0.05) reduced the lesion area of fruits inoculated with Trichothecium roseum and the natural disease incidence of fruits during storage at room temperature. The treatment effectively maintained cell membrane integrity and decreased the production of lipid peroxidation and malondialdehyde. BTH treatment markedly enhanced the accumulation of hydrogen peroxide, (H2O2) while decreased the rate of superoxide anion (O2·−) production. Furthermore, BTH observably increased the activities of NADPH oxidase (NOX, EC1.6.3.1), superoxide dismutase (SOD, EC1.15.1.1), peroxidase (POD, EC1.11.1.7), ascorbate peroxidase (APX, EC1.11.1.11), and glutathione reductase (GR, EC1.6.4.2), while inhibited the catalase (CAT, EC1.11.1.6) activity. These results indicated that BTH treatment increased the disease resistance of muskmelon fruits by enhancing their antioxidant system activity after harvest, and suggested that postharvest treatment with BTH could be promising in reducing decay and reducing or/and substituting chemical fungicides to control postharvest diseases in fruits.

Coptisine exert cardioprotective effect through anti-oxidative and inhibition of RhoA/Rho kinase pathway on isoproterenol-induced myocardial infarction in rats

ObjectiveBecause myocardial infarction is a major cause of morbidity and mortality worldwide, protecting the heart from the ischemia is the focus of intense research. Coptisine is an isoquinoline alkaloid extracted form Coptidis Rhizoma. This study aims to elucidate if coptisine is responsible for cardioprotection using myocardial infarction (MI) rat models and investigate its potential mechanism of action. MethodsMyocardial infarction was produced in rats with 85mgkg−1 isoproterenol administered subcutaneously twice at an interval of 24h. The rats were randomized into 7 groups: (I) Normal; (II) ISO; (III) ISO+fasudil; (IV) ISO+isosorbide dinitrate (ISDN) and (V–VII) ISO+coptisine (25, 50 and 100mgkg−1). Cardiac function and markers of cardiac ischemic were assessed after MI. ResultsRats pretreated with coptisine (25, 50 and 100mgkg−1) for 21 days and received subcutaneously injected with ISO (85mgkg−1) on the 20th and 21st day at an interval of 24h. The results suggested that coptisine has strong antioxidant activity, and it can maintain cell membrane integrity, ameliorate mitochondrial respiratory dysfunction, reduce myocardial cells apoptosis, inhibit RhoA/ROCK expression induced by high-dose isoproterenol administration. ConclusionsCoptisine provided cardioprotection in a model of myocardial infarction, and therefore should be considered as a novel adjunctive therapy for attenuating myocardial damage.

Impact of 1-MCP on postharvest quality of sweet cherry during cold storage

Sweet cherry (Prunus avium L.) is a kind of fruit with short postharvest life. Postharvest treatment of 1-methylcyclopropene (1-MCP: 1.0, 1.5 and 2.0 μL/L) was applied to cherry fruits, and its effects on postharvest fruit quality during 60-day-storage at cold temperature was investigated. Sweet cherry fruits were harvested at commercial fruit maturity and exposed to 1-MCP at 20±1°C for 24 h. Following 1-MCP treatments, the fruits were stored for 60 d at 90±5% RH and, 0±1°C, in three concentrations of 1.0, 1.5, and 2.0 μL/L 1-MCP, of which 1.5 μL/L 1-MCP had the most positive effect on fruit quality. Postharvest treatments of 1-MCP significantly reduced endogenous ethylene production, malondialdehyde (MDA) content, and polyphenol oxidase (PPO) activity in cherry fruits 60 d after cold storage when compared to untreated fruits. 1-MCP treatment was associated with lower respiration rate and relative electric conductivity (REC) and maintained cell membrane integrity. The results indicated that the treatment 1-MCP was effective in inhibition of the declining of POD activity and CATactivity. In conclusion, 1-MCP treatment could be a good candidate for maintaining postharvest quality of cherry, and 1-MCP could prolong the storage life of sweet cherry.

Requirement of a Tsp2-Type Tetraspanin for Laccase Repression and Stress Resistance in the Basidiomycete Cryptococcus neoformans

Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.

Apoptotic cell death “Nixed” by an ER–mitochondrial necrotic pathway

Cells commit suicide in a highly regulated manner to sculpt an organism during development, to maintain homeostasis in postnatal tissues, and in response to noxious cues. It was thought until recently that cells in this situation were eliminated solely by apoptosis, a process whereby they shrink, fragment, and undergo phagocytosis—all while maintaining plasma membrane integrity and thereby avoiding inflammation. Necrosis, a nonapoptotic form of cell death characterized by depletion of cellular ATP, plasma membrane rupture, cell swelling, and marked inflammation, has been recognized for centuries but until recently was considered a passive and unregulated process. Work in worms and mammals over the past decade, however, has led to the unexpected discovery that, at least in some instances, necrosis can be deliberate and regulated (1–5). Although intensive work over the past 20 years has produced a mature blueprint for the molecular regulation of apoptosis (6), delineation of programmed necrosis signaling remains a work in progress. Nevertheless, an obvious next step is to begin to elucidate the interface between molecular events that mediate apoptosis and necrosis. As reported in PNAS, Chen et al. (7) manipulated a protein that can induce cell death with either apoptotic or necrotic features to probe the relationship between apoptosis and necrosis mechanisms in the mitochondria and endoplasmic reticulum (ER).

Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma.

Abstract 113 Background:Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether. Methods:To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC50 for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling. Results:Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC50 values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC50 of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC50. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide. Conclusions:These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo. Disclosures:No relevant conflicts of interest to declare.