Membrane Aggregation Research Articles

Flavone-Rich Fractions and Extracts from Oroxylum indicum and Their Antibacterial Activities against Clinically Isolated Zoonotic Bacteria and Free Radical Scavenging Effects.

Oroxylum indicum extracts from the seeds collected from Lampang and Pattani provinces in Thailand, and young fruits and flowers exhibited in vitro display antioxidant and antibacterial activities against clinically isolated zoonotic bacteria including Staphylococcus intermedius, Streptococcus suis, Pseudomonas aeruginosa, β-hemolytic Escherichia coli and Staphylococcus aureus. The orange crystals and yellow precipitates were obtained from the preparation processes of the seed extracts. The orange-red crystals from the seeds collected from Lampang province exhibited strong in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging effects (EC50 value = 25.99 ± 3.30 μg/mL) and antibacterial effects on S. intermedius and β-hemolytic E. coli while the yellow precipitate from the same source exhibited only antioxidant activity. Quantitative analysis of phytochemicals in O. indicum samples by spectrophotometric and HPLC techniques showed that they contained different amounts of total phenolic, total flavonoid and three major flavones; baicalin, baicalein and chrysin contents. Young fruit extract, which contained low amounts of flavone contents, still promoted antibacterial effects against the tested bacteria with IC50 values lower than 1 mg/mL and MIC values between 4 to 10 mg/mL in S. intermedius, S. aureus and S suis while higher IC50 and MIC values against P. aeruginosa and β-hemolytic E. coli were found. From scanning electron microscopy, the extract of the young fruit of O. indicum promoted morphological changes in the bacterial cells by disrupting the bacterial cell walls, inducing leakage of the cellular content, and generating the abnormal accumulation of cells. The mechanism of action of the extract for this antibacterial effect may be the disruption of the cell membrane and abnormal cell aggregations. Regression analysis of the results suggests the correlation between total phenolic and total flavonoid contents and antioxidant and antibacterial effects. Baicalin was found to have a high correlation with an inhibitory effect against β-hemolytic E. coli while three unidentified peaks, which could be flavones, showed high correlations with an inhibitory effect against S. intermedius, S. suis, P. aeruginosa and S. aureus.

Nitrogen-rich graphene aerogel with interconnected thousand-layer pancake structure as anode for high performance of lithium-ion batteries

Nitrogen-rich graphene aerogel with interconnected thousand-layer pancake structure (NTLP) was designed and fabricated through hydrothermal template self-assembly method using polystyrene (PS) nanospheres as structural guiding templates and ethylenediamine (EDA) as a nitrogen resource for in-situ N-doping. NTLP exhibits significantly improved electrochemical performance of lithium-ion storage including a highly reversible capacity of 1147 ​mA ​h g−1 at 0.1 ​A ​g−1, and favorable rate capability of 258 ​mA ​h g−1 even at a large current density of 5 ​A ​g−1. Moreover, the reversible capacity is retained at 501 ​mA ​h g−1 after 1100 cycles at 1 ​A ​g−1. PS template effectively alleviates the aggregation of graphene membrane, meanwhile forms peculiar thousand-layered cake structure with high specific surface area and well-controlled pores. Precisely owing to the unique structure with three-dimensionally (3D) interconnected holey graphene network and the introduction of abundant N-doping (about 7 atom%) into the graphene walls, NTLP significantly enhances the electrochemical activity and effectively accelerates the diffusions of both lithium-ions and electrons. The high performance strongly indicates that NTLP possesses a promising perspective for lithium-ion batteries with excellent electronic conductivity, efficient transport pathways of electrons and ions as well as superior reversible lithium storage capacity.

Molecular Dynamics Simulations Reveal Membrane Interactions for Poorly Water-Soluble Drugs: Impact of Bile Solubilization and Drug Aggregation

Molecular transport mechanisms of poorly soluble hydrophobic drug compounds to lipid membranes were investigated using molecular dynamics (MD) simulations. The model compound danazol was used to investigate the mechanism(s) by which bile micelles delivered it to the membrane. The interactions between lipid membrane and pure drug aggregates—in the form of amorphous aggregates and nanocrystals—were also studied. Our simulations indicate that bile micelles formed in the intestinal fluid may facilitate danazol incorporation into cellular membranes through two different mechanisms. The micelle may be acting as: i) a shuttle that presents the danazol directly to the membrane or ii) an elevator that moves the solubilized danazol with it as the colloidal structure itself becomes incorporated and solubilized within the membrane. The elevator hypothesis was supported by complementary lipid monolayer adsorption experiments. In these experiments, colloidal structures formed with simulated intestinal fluid were observed to rapidly incorporate into the monolayer. Simulations of membrane interaction with drug aggregates showed that both the amorphous aggregates and crystalline nanostructures incorporated into the membrane. However, the amorphous aggregates solubilized more quickly than the nanocrystals into the membrane, thereby improving the danazol absorption.

Investigation of epididymal proteins and general sperm membrane characteristics of Formosan pangolin (Manis pentadactyla pentadactyla)

BackgroundFormosan Pangolin (Manis pentadactyla pentadactyla) is one of the three subspecies of Chinese pangolins, it is also an isolated sub-species naturally habitat in Taiwan. Despite earlier report on successful breeding of Sunda (Manis javanica) pangolin, breeding of Formosan pangolins in zoo captive populations is still challenging due to unknown reproductive characterizations of this species in both male and female populations.ResultsWe characterized for the first time, reproductive tract of male Formosan pangolin. We showed pangolin epididymis was a collagen-enriched organ with apparent segmented sub-regions similar to other mammals. However, unlike most mammals exhibited two V-ATPase subunits, Formosan pangolin exhibited only V-ATPase subunit 2. This specific V-ATPase subunit extended its cellular localization throughout the cytoplasm of epididymal clear cells, suggesting pH regulation of luminal microenvironment might be different from other mammals. Electron micrographs showed rod-shaped pangolin sperm cells with multi-lamellar membrane structure at the sperm head. Similar to well-defined capacitation and acrosome reaction membrane changes in other mammals, we reported three distinct patterns (homogenous, punctuated and faded) of pangolin sperm head membrane changes. The concurrent increase in phosphotyrosine protein expression detected at the sperm mid-piece/tail and the emergence of punctuated membrane aggregates likely representing three sperm activation stages, namely inactivated, capacitated and acrosome reacted status of pangolin sperm.ConclusionBy revealing unique epididymal V-ATPase distribution and sperm membrane dynamics in Formosan pangolin, we would understand better the fundamental aspects of reproduction parameters of Formosan pangolin.

The Effects of Resveratrol, Caffeine, β-Carotene, and Epigallocatechin Gallate (EGCG) on Amyloid- Aggregation in Synthetic Brain Membranes.

Alzheimer's disease is a neurodegenerative condition marked by the formation and aggregation of amyloid-β (Aβ) peptides. There exists, to this day, no cure or effective prevention for the disease; however, there is evidence that a healthy diet and certain food products can slow down first occurrence and progression. To investigate if food ingredients can interact with peptide aggregates, synthetic membranes that contained aggregates consisting of cross-β sheets of the membrane active fragment A areprepared. The impact of resveratrol, found in grapes, caffeine, the main active ingredient in coffee, β-carotene, found in orange fruits and vegetables, and epigallocatechin gallate (EGCG), a component of green tea, on the size and volume fraction of Aβ aggregates is studied using optical and fluorescence microscopy, X-ray diffraction, UV-vis spectroscopy, and molecular dynamics simulations. All compounds are membrane active and spontaneously partitioned in the synthetic brain membranes. While resveratrol and caffeine lead to membrane thickening and reduced membrane fluidity, β-carotene and EGCG preserve or increasefluidity. Resveratrol and caffeine do not reduce the volume fraction of peptide aggregates while β-carotene significantly reduces plaque size. Interestingly, EGCG dissolves peptide aggregates and significantly decreases the corresponding cross-β and β-sheet signals.

The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Peroxisome Proliferator Activator Receptor Gamma Coactivator-1α Overexpression in Amyotrophic Lateral Sclerosis: A Tale of Two Transgenics.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder manifesting with upper and lower neuron loss, leading to impairments in voluntary muscle function and atrophy. Mitochondrial dysfunction in metabolism and morphology have been implicated in the pathogenesis of ALS, including atypical oxidative metabolism, reduced mitochondrial respiration in muscle, and protein aggregates in the mitochondrial outer membrane. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays an essential role in the regulation of mitochondrial biogenesis, the process by which existing mitochondria grow and divide. PGC-1α has been previously reported to be downregulated in the spinal cord of individuals with ALS. Towards targeting PGC-1α as a therapeutic mechanism, we have previously reported improved motor function and survival in the SOD1G93A mouse model of ALS by neuron-specific over-expression of PGC-1α under a neuron-specific enolase (NSE) promoter. As pharmacological intervention targeting PGC-1α would result in whole-body upregulation of this transcriptional co-activator, in the current study we investigated whether global expression of PGC-1α is beneficial in a SOD1G93A mouse model, by generating transgenic mice with PGC-1α transgene expression driven by an actin promoter. Actin-PGC-1α expression levels were assayed and confirmed in spinal cord, brain, muscle, liver, kidney, and spleen. To determine the therapeutic effects of global expression of PGC-1α, wild-type, actin-PGC-1α, SOD1G93A, and actin-PGC-1α/SOD1G93A animals were monitored for weight loss, motor performance by accelerating rotarod test, and survival. Overexpression of actin-PGC-1α did not confer significant improvement in these assessed outcomes. A potential explanation for this difference is that the actin promoter may not induce levels of PGC-1α relevant to disease pathophysiology in the cells that are specifically relevant to the pathogenesis of ALS. This evidence strongly supports future therapeutic approaches that target PGC-1α primarily in neurons.

Experimental estimation of the effects of exogenous carbon monoxide on blood cells

The aim of the study was to investigate the effect of the carbon monoxide (CO) donor on the Ca 2+ -activated potassium permeability of the erythrocyte membrane and platelet aggregation ability. Materials and methods. Healthy volunteers ( n = 27) and patients with chronic coronary heart disease (CHD) ( n = 32) of both sexes were examined. The material of the study was packed red blood cells and platelet-rich plasma obtained from patient’s venous blood. The change of Ca 2+ -dependent potassium conductivity of the erythrocyte membrane was evaluated by potentiometric method, and the platelet aggregation was studied by turbidimetric method. Carbon monoxide releasing molecule-2 ( CORM-2) was used as a CO donor. The amplitude of A23187- and redox-induced hyperpolarization response (HR) of erythrocytes, and the rate and degree of platelet aggregation were estimated. Results. It was shown that the addition of CORM-2 (10 and 100 μM) in the erythrocyte suspension caused a dose-dependent decrease in the amplitude of A23187- and redox-dependent HR in healthy donors, as well as in patients with chronic CHD. The maximum decrease was observed in the presence of 100 μM CORM-2. The effect of CORM-2 at concentrations of 10 and 100 μM on collagen-induced platelet aggregation led to a decrease in the degree and rate of aggregation in healthy donors. The maximum effect was shown at 100 μM of CO donor. However, such an unambiguous effect of CORM-2 on the aggregation parameters in patients with CHD was not observed. Conclusion . The results suggest that CO has a significant effect on the ion transport function of the erythrocyte membrane and platelet aggregation activity of both healthy donors and patients with CHD.

Annexin A1 in Malignancy

Annexin A1 (Anxa1) is an agent of the superfamily of Annexin proteins. Previously, it was known as a mediator in the anti-inflammatory response of glucocorticoids. In several types of research in past decades, it was reported as lipomodulin, lipocortin-1, renocortin, and macrocortin, before it was termed Annexin A1. Anxa1 has complex roles in different essential cellular processes as membrane aggregation, cellular transduction, phagocytosis, inflammation, differentiation, proliferation, and apoptosis. Cumulative evidence has suggested that Anxa1 dysregulations are linked to tumorigenesis, tumor progression, metastasis, invasion, and drug resistance of cancers by contributing to cell proliferation and differentiation, cell apoptosis and signaling. Recently, there is growing evidence indicates that Anxa1 could function either as a tumor promoter or a tumor suppressor candidate for certain cancers relying on the specific type of tumor cells/tissues.In this article, we attempt to clarify the association between Annexin A1 and malignancies, as well as potential action mechanisms. Anxa1 may serve as prognostic predictor of outcome and response to chemotherapy and a potential target for therapy in the future.

COMPARISON OF THE EFFECTIVENESS BETWEEN RED GALANGAL RHIZOME'S (Alpinia purpurata k.schum) EXTRACT AND KETOCONAZOL 2% TO INHIBIT GROWTH OF DERMATOPHYTE ​​FUNGI IN VITRO

Red galangal rhizome (Alpinia purpurata k. Scum) has a larger size and has more therapeutic properties. Red galangal has compounds that can inhibit fungal growth namely flavonoids and saponins which can interfere with the function of the fungal cytoplasmic membrane, besides that they can cause leakage and aggregation of the outer cell membrane, which can inhibit the growth of dermatophytes. This study is a laboratory experimental study, using antifungal activity test with diffusion method using paper discs to see the comparison of the red galangal extract effectiveness and ketoconazole 2% on the growth of dermatophytes fungi Microsporum canis, Microsporum gypseum, and Trichophyton metagrophytes by measuring the inhibition zone around the fungal colonies. The concentrations of red galangal extract used were 10%, 20%, 30%, 40%, and 50% and used 2% ketoconazole as control. The data was analyzed using the One Way Anova test. Result of this study shown that all concentrations without 10 % of red galangal have inhibited zones of Microsporum canis, Microsporum gypseum, and Trichophyton metagrophytes but not better than ketoconazole 2 % activities.

Membrane Disruption and Peptide/Lipid Co-Assembly by the Amyloid-Forming Peptide, PAP248-286

Both amyloid-forming and antimicrobial peptides (AMPs) are membrane active, displaying a variety of membrane-disrupting and -remodeling activities including membrane leakage, vesicle fusion, and vesicle aggregation. The cytotoxicity of membrane disrupting pre-fibrillar oligomers underlies the pathological activity of amyloid-forming peptides. Conversely, the disruption and aggregation of microbial membranes by AMPs is critical to their function in an innate immune response. Therefore, studying the factors driving the membrane activity of these peptides may be valuable in understanding both normal function and diseased states. We focus our studies on an amyloidogenic fragment of prostatic acid phosphatase (PAP248-286). In its amyloid state, this peptide is termed “semen-derived enhancer of viral infection” (SEVI), and greatly increases HIV infectivity. It is also thought to play functional roles in the antimicrobial activity of seminal fluid and sperm clearance during fertilization. Using liposomes as model membranes and biophysical tools including negative stain electron microscopy, dynamic light scattering, and single particle fluorescence burst analysis, we have demonstrated that PAP248-286 displays dramatic membrane remodeling activities, as it can form large, fibrillar lipid-peptide co-aggregates that are distinct from SEVI amyloid and form much more readily. We have also found that PAP248-286 is a potent membrane disruptor. Using fluorescence dequenching experiments we have determined that PAP248-286 disrupts liposomes in a charge and dose dependent manner. We have also developed new approaches to improve the fitting and analysis of complex time-correlated single photon counting (TCSPC) experimental data, which has allowed us to characterize its leakage mechanism. Our work to elucidate the determinants of PAP248-286 membrane activity is not only important for understanding its biological activities, but also provides a framework for studying the membrane activity of other biologically important amyloid-forming and antimicrobial peptides.

Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.

Autophagy plays an essential role in the defense against many microbial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1β (IL-1β). These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.

PATTERN OF ACTIVE BLOOD DONORS DONATING FOR MORE THAN 10 YEARS BASED ON THE RESULTS OF LABORATORY, MORPHOLOGIC, BIOCHEMICAL AND BIOPHYSICAL TESTS OF PERIPHERAL BLOOD

Introduction: In spite of the lately increasing number of scientific research programs on donor blood storage, integrated solution of this problem remains a challenging open issue. The aim: upon the study of laboratory, morphologic, biochemical and biophysical properties of donor blood erythrocytes, determine pathogenesis of abnormalities in erythrocytes of blood donors depending and increase effectiveness of early diagnostics and prevention of the above changes for donors' health protection. Materials and methods: 215 blood donors (112 men and 103 women) were examined, among which 55 were active donors (29 men and 26 women) donating on regular basis, no less than 3 times a year and 160 first-time registered donors (83 men and 77 women). First-time registered donors made the control group of our research. The following methods were used: general blood tests, blood chemistry, radioimmune and enzyme-immunoassay, statistic methods. Results: Results of the performed laboratory, morphologic, biochemical and biophysical tests: erythrocyte index determination, reticulocyte count, red blood cell distribution width, optical transmission of erythrocytes, test for aggregation and penetrability of erythrocyte membranes, effectiveness of erythropoiesis value determination in regular donor's and first-time donors erythrocytes demonstrated that the examined person making the group of donors are practically healthy people whose test results are within normal limits for their age group. Conclusions: In order to preserve health of donors and ensure quality of blood components received at the time of donation, thorough checkup of donors, including, apart from the main and biochemical peripheral venous blood parameters, morphologic, biophysical and rheological parameters of erythrocytes is highly recommended before donation.

Trema orientalis (Linn.) leaves promotes anticancer activity in Ehrlich ascites carcinoma (EAC) in Swiss albino mice.

Background The in vivo anticancer effect of the Trema orientalis leaves crude methanol extract (TLME) was screened against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. Materials and methods The cytotoxic activity of TLME was determined in vitro by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The growth inhibitory activity and morphological alterations were determined by the hemocytometer counting of the EAC cells using trypan blue dye. The apoptotic cells were assessed by DAPI (4',6-diamidino-2-phenylindole) staining. The hematological and biochemical parameters of experimental mice were also estimated. Results After treatment with the TLME, the viable tumor cell count, morphological changes and nuclear damages of the EAC cells were observed along with the hematological parameters of the experimental mice. The LD50 of TLME was 3120.650 mg/kg body weight, and this extract was proven to be safe at a dose of as high as 800 mg/kg body weight. The oral administration of the TLME at 400 mg/kg body weight resulted in approximately 59% tumor cell growth inhibition compared with the control mice, with considerable apoptotic features, including membrane blebbing, chromatin condensation, nuclear fragmentation and aggregation of the apoptotic bodies in DAPI staining under a fluorescence microscope. The TLME also dose-dependently restored the altered hematological parameters to approximately normal levels. The TLME exhibited bolstering cytotoxic effect against the EAC cell with the IC50 value of 29.952 ± 1.816 μg/mL. Conclusion The TLME has potential as a natural anti-cancer product with apoptosis induction property and cytotoxicity against carcinoma cells.

Effect of lipid saturation on amyloid-beta peptide partitioning and aggregation in neuronal membranes: molecular dynamics simulations

Aggregation of amyloid-beta (Aβ) peptides, cleaved from the amyloid precursor protein, is known as a precursor of the Alzheimer’s disease (AD). It is also known that Alzheimer’s disease is characterized by a substantial decrease of the amount of polyunsaturated lipids in the neuronal membranes of the frontal gray matter. To get insight into possible interconnection of these phenomena, we have carried out molecular dynamics simulations of two fragments of Abeta peptide, Abeta _{1-28} and Abeta _{26-40}, in four different lipid bilayers: two monocomponent ones (14:0-14:0 PC, 18:0-22:6 PC), and two bilayers containing mixtures of 18:0-18:0 PE, 22:6-22:6 PE, 16:0-16:0 PC and 18:1-18:1 PC lipids of composition mimicking neuronal membranes in a “healthy” and “AD” brain. The simulations showed that the presence of lipids with highly unsaturated 22:6cis fatty acids chains strongly affects the interaction of amyloid-beta peptides with lipid membranes. The polyunsaturated lipids cause stronger adsorption of Abeta -peptides by the membrane and lead to weaker binding between peptides when the latter form aggregates. This difference in the behaviour observed in monocomponent bilayers is propagated in a similar fashion to the mixed membranes mimicking composition of neuronal membranes in “healthy” and “AD” brains, with “healthy” membrane having higher fraction of polyunsaturated lipids. Our simulations give strong indication that it can be physical–chemical background of the interconnection between amyloid fibrillization causing Alzheimer’s disease, and content of polyunsaturated lipids in the neuronal membranes.

Structure of Membrane-Bound Huntingtin Exon 1 Reveals Membrane Interaction and Aggregation Mechanisms

Huntington's disease is caused by a polyQ expansion in the first exon of huntingtin (Httex1). Membrane interaction of huntingtin is of physiological and pathological relevance. Using electron paramagnetic resonance and Overhauser dynamic nuclear polarization, we find that the N-terminal residues 3-13 of wild-type Httex1(Q25) form a membrane-bound, amphipathic α helix. This helix is positioned in the interfacial region, where it is sensitive to membrane curvature and electrostatic interactions with head-group charges. Residues 14-22, which contain the first five residues of the polyQ region, are in a transition region that remains in the interfacial region without taking up a stable, α-helical structure. The remaining C-terminal portion is solvent exposed. The phosphomimetic S13D/S16D mutations, which are known to protect from toxicity, inhibit membrane binding and attenuate membrane-mediated aggregation of mutant Httex1(Q46) due to electrostatic repulsion. Targeting the N-terminal membrane anchor using post-translational modifications or specific binders could be a potential means to reduce aggregation and toxicity invivo.

Characterization of TNF-induced cell death in Drosophila reveals caspase- and JNK-dependent necrosis and its role in tumor suppression

Tumor-necrosis factor (TNF) and its superfamily members are pleiotropic cytokines. Activation of TNF can lead to distinct cellular outcomes including inflammation, cell survival, and different forms of cell death, such as apoptosis and necrosis in a context-dependent manner. However, our understanding of what determines the versatile functions of TNF is far from complete. Here, we examined the molecular mechanisms that distinguish the forms of cell death induced by Eiger (Egr), the sole homolog of TNF in Drosophila. We show that expression of Egr in the developing Drosophila eye simultaneously induces apoptosis and apoptosis-independent developmental defects indicated by cellular disorganization, both of which rely on the c-Jun N-terminal kinase (JNK) signaling activity. Intriguingly, when effector caspases DrICE and Dcp-1 are defective or inhibited, expression of Egr triggers necrosis which is characterized by loss of cell membrane integrity, translucent cytoplasm, and aggregation of cellular organelles. Moreover, such Egr-induced necrosis depends on the catalytic activity of the initiator caspase Dronc and the input from JNK signaling but is independent of their roles in apoptosis. Further mosaic analysis with mutants of scribble (scrib), an evolutionarily conserved tumor suppressor gene regulating cell polarity, suggests that Egr/JNK-mediated apoptosis and necrosis establish a two-layered defense system to inhibit the oncogenic growth of scrib mutant cells. Together, we have identified caspase- and JNK-dependent mechanisms underlying Egr-induced apoptosis versus necrosis and their fail-safe roles in tumor suppression in an intact organism in vivo.

Hypoxic storage of erythrocytes slows down storage lesions and prolongs shelf-life.

Conventional storage conditions of erythrocytes cause storage lesions. We propose that hypoxic storage conditions, involving removal of oxygen and replacement with helium, the changes in stored erythrocytes under hypoxic condition were observed and assessed. Erythrocytes were divided into two equal parts, then stored in conventional and hypoxic conditions, separately. Blood gas analysis, hemorheology, and hemolysis were performed once a week. Energy metabolism and membrane damage were monitored by enzyme-linked immunosorbent assay. Phosphatidylserine exposure was measured by flow cytometry. P50 was measured and the oxygen dissociation curve(ODC) plotted accordingly. Erythrocyte morphology was observed microscopically. In the 9th week of storage, the hemolysis of the hypoxia group was 0.7%; lower (p < .05) than that of the control group and still below the threshold of quality requirements. The dissolved oxygen and pO2 were only 1/4 of that in the control group (p < .01); the adenosine triphosphate, glucose, and lactic acid levels were decreased (p < .05), while the 2,3-diphosphoglycerate levels were increased relative to that in the control group (p < .01). There were no statistically significant differences in membrane damage, deformability, and aggregation between the two groups. In addition, the ODC of the two groups was shifted to the left but this difference was not statistically different. Basically similar to the effect of completely anaerobic conditions. Erythrocytes stored under hypoxic conditions could maintain a relatively stable state with a significant decrease in hemolysis, reduction of storage lesions, and an increase in shelf-life.

Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition.

Microcavity-supported lipid bilayers (MSLBs) are contact-free membranes suspended across aqueous-filled pores that maintain the lipid bilayer in a highly fluidic state, free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer a significant opportunity to model membrane asymmetry, protein-membrane interactions, and aggregation at the membrane interface. Herein we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity, and cholera toxin-ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phases, and ternary phase-separated membrane compositions. Fluorescence lifetime correlation spectroscopy was used to determine the lateral mobility of the lipid and the protein, and electrochemical impedance spectroscopy enabled the detection of the protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have a profound impact on membrane electrochemical resistance, where the resistance of a ternary symmetric phase-separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition in the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 times faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that the interplay of lipid packing across both membrane leaflets and the concentration of GM1 both affect the extent of cholera toxin aggregation and the consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity to build greater biomimicry into biophysical models, and the approach described demonstrates the value of MSLBs in studying aggregation and the membrane-associated multivalent interactions prevalent in many carbohydrate-mediated processes.

How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency.

Organisms that thrive at cold temperatures produce ice-binding proteins to manage the nucleation and growth of ice. Bacterial ice-nucleating proteins (INP) are typically large and form aggregates in the cell membrane, while insect hyperactive antifreeze proteins (AFP) are soluble and generally small. Experiments indicate that larger ice-binding proteins and their aggregates nucleate ice at warmer temperatures. Nevertheless, a quantitative understanding of how size and aggregation of ice-binding proteins determine the temperature Thet at which proteins nucleate ice is still lacking. Here, we address this question using molecular simulations and nucleation theory. The simulations indicate that the 2.5 nm long antifreeze protein TmAFP nucleates ice at 2 ± 1 °C above the homogeneous nucleation temperature, in good agreement with recent experiments. We predict that the addition of ice-binding loops to TmAFP increases Thet, but not enough to compete in efficiency with the bacterial INP. We implement an accurate procedure to determine Thet of surfaces of finite size using classical nucleation theory, and, after validating the theory against Thet of the proteins in molecular simulations, we use it to predict Thet of the INP of Ps. syringae as a function of the length and number of proteins in the aggregates. We conclude that assemblies with at most 34 INP already reach the Thet = -2 °C characteristic of this bacterium. Interestingly, we find that Thet is a strongly varying nonmonotonic function of the distance between proteins in the aggregates. This indicates that, to achieve maximum freezing efficiency, bacteria must exert exquisite, subangstrom control of the distance between INP in their membrane.