Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.

The investigation for the relationship among serum leptin, erythrocyte membrane Ca^(2+)-ATPase activity and hypertensive disorder complicating pregnancy

Objective： To study the significance of Leptin and the activity of erythrocyte membrane Ca^2＋-ATPase（EMCA） in the development of hypertensive disorder complicating pregnancy. Methods： Radioimmunoassay was used to test the level of serum Leptin, and the activity of EMCA was determined chemically in 38 pregnant women with hypertensive disorder complicating pregnancy and 36 normotensive pregnant women. Results： The level of serum Leptin in hypertensive disorder complicating pregnancy（gestational hypertension： 13.76 ± 3.46 ng/ml; preeclampsia： 15.76 ± 5.47 ng/ml; eclampsia： 18.32 ± 6.38 ng/ml）was significantly higher than that in normotensive pregnant women （11.33 ± 2.93 ng/ml） ,respectively. The average EMCA activity of patients with hypertensive disorder complicating pregnancy （gestational hypertension： 1.65 ± 0.24 μmol·pi/mg.h ; preeclampsia： 1.37 ± 0.19 μ mol·pi/mg·h;eclampsia：1.12 ± 0.14 μmol·pi/mg·h） was significantly lower than that of normotensive pregnant women（1.83 ± 0.38 μ mol·pi/mg·h）,respectively. There was a negative correlation between the level of serum Leptin and the activity of RMCA in hypertensive disorder complicating pregnancy （r = -0.63）. Conclusion： Inhibition of EMCA activity of erythrocyte in hypertensive disorder complicating pregnancy may increase cytoplasmic free calcium, which contributes to the development of hypertensive disorder complicating pregnancy. The negative correlation between the level of serum Leptin and the activity of EMCA, also suggested that serum Leptin and the activity of EMCA may play a role in the development of hypertensive disorder complicating pregnancy.

Elementary study on the antigenicity of SARS-CoV Mc region

To test the antigenic activity of M protein （Mc protein） in the inner membrane of SARS-CoV, SARS-CoV Me protein＇s bases locating inside the membrane were cloned, the His-fusion protein was expressed in E. coli and analyzed for its antigenic activity. Among those 7 clinically diagnosed patients＇ sera, there were 5 positive and 2 negative in reaction with His-fusion protein. All of the 20 healthy persons＇ sera and rabbit anti-OC43 and 229E were of negative reaction with His-fusion protein. The animals immunized with His-fusion protein have produced muhi-clonal antibody. The His-fusion protein could specially react with clinically diagnosed SARS patients＇ sera and the animals immunized with His-fusion protein could produce specifically multi-clonal antibody, but it could not react with the sera of healthy persons and the rabbit anti-OC43 and 229E.

Mixed Matrix Membranes of Polysulfone/Polyimide Reinforced with Modified Zeolite Based Filler: Preparation, Properties and Application

In this work, polysulfone/polyimide（PSf/PI） mixed matrix membranes were fabricated by reinforcement of modified zeolite（MZ） particles through solution casting method for investigation of antibacterial activity against two gram negative bacteria（Salmonella typhi, Klebsella pneumonia） and two gram positive bacteria（Staphylococcus aureus, Bacillus subtilis）. The modified zeolite particles were incorporated to PSf and PI matrix and the influence of these particles on thermal, mechanical and structural properties was evaluated. The morphological evolution was investigated through scanning electron microscopy（SEM） and transmission electron microscopy（TEM） analysis, which revealed good compatibility between organic polymer matrix and inorganic filler. Mechanical stability was investigated by tensile testing while thermal analysis was evaluated by thermogravimetric analysis（TGA） and differential scanning calorimetry（DSC）. This revealed improvement in thermal properties with increasing filler concentration from 1 wt% to 10 wt%. Structural analysis was successfully done using X-ray diffraction analysis（XRD） and Fourier transform infrared（FTIR） spectroscopy. Solvent content of fabricated mixed matrix membranes was observed to decrease while moving from more hydrophilic to less hydrophilic solvent. However, addition of filler content enhanced the porosity of fabricated membranes. The synthesized mixed matrix membranes exhibited good antibacterial activity and the highest activity was shown by PSf/PI/MZ mixed matrix membrane. Therefore, the combination effect of PSf, PI and MZ sufficiently enhanced the antibacterial activity of mixed matrix membranes.

Na^+-K^+-ATPase activity of hepatocyte plasma membranes,reversible and irreversible alterations due to bile acids

Background Chenodeoxycholic acid (CDC) is an apolar bile salt and damages hepatocytes, whereas ursodeoxycholic acid (UDC) is a more polar bile salt and protects liver cell against toxic bile salts. We therefore investigated the activity of membrane associated Na + K + ATPase, a coenzyme of the sodium dependent bile salt carrier. Methods Liver plasma membranes (LPM) were isolated from rat livers according to the method of Song et al (J Cell Biol 1969; 41:124). The LPM were incubated with bile salts (TUDC, UDC, TCDC, CDC) in concentrations of 0.1 2 mmol/L for 0 30 minutes at room temperature. To study reversibility of the effect of CDC, LPM were diluted with buffer 50 times of volume after incubation. The activity of membrane associated Na + K+ ATPase was determined enzymatically at 37℃ and the phospholipid (PL) release into the supernatant was measured. Results CDC and TCDC both showed a dose dependent inhibition of the enzyme activity (P<0.01 vs control). Gastroenterology, Center of Internal Medicine, University Hospital, D 60590 Frankfurt Main, Germany (You T, Guldutuna S, Bhatti S and Leuschner U)Initially TCDC induced an increase in enzyme activity at concentrations of 0.1 1 mmol/L, however, after 3 minutes activity repidly decreased to less than 30% of controls. Up to a concentration of 1 mmol/L CDC the inhibition of enzyme activity could be reversed by diluting the bile salt in the incubation medium. At a concentration of 2 mmol/L CDC activity was only partially restored and at this concentration a marked PL release into the supernatant was observed, indicating solubilization of the membranes. UDC did not decrease the enzyme activity at concentrations of 0.1 2 mmol/L. At a concentration of 2 mmol/L TUDC inhibited the Na + K + ATPase by about 20%. Solubilization of membrane PL was not observed. Conclusion CDC and TCDC inhibited Na + K + ATPase. Dilution of the bile salt with buffer reversed the inhibitory effect up to concentrations of 1 mmol/L CDC. The inhibitory effect is probably due to alteration of the plasma membrane. 2 mM CDC caused irreparable membrane damage. Physiological concentrations of UDC and TUDC did not affect membrane ATPase.

Structural engineering of antimicrobials for optimal broad-spectrum activity

Antimicrobial materials are a crucial component in eradicating and managing the spread of infectious diseases.They are expected to act on a broad-spectrum of microbes,including emerging pathogens which could cause the next Disease X.Herein,we reassessed a series of antimicrobial imidazolium polymers on our shelves and uncovered extended functionality through dual modes of action.By redesigning their structures,a truly broadspectrum antimicrobial material with optimized activity against bacteria(G+ve,G-ve)and fungi,as well as enveloped and non-enveloped viruses was developed.We demonstrated that the imidazolium polymer exhibits dual modes of function against microbes:targeting the microbial membrane and binding DNA.The latter DNA binding affinity was found to be key against non-enveloped viruses.With this insight,we designed small molecule compounds that exhibited optimum broad-spectrum antimicrobial activity and excellent efficacy against ESKAPE group of pathogens that are responsible for some of the deadliest nosocomial infections worldwide.Our results could also shed light on the design of broad-spectrum antimicrobial compounds against Disease X.

Accumulation of ciprofloxacin and lomefloxacinin fluoroquinolone-resistant strains of Escherichia coli

Objective To evaluate the role of outer membrane protein (Omp) F-deficiency and active efflux in the accumulation of hydrophilic fluoroquinolones ciprofloxacin (CPLX) and lomefloxacin (LMLX) in resistant E. coli strains. Methods Fluoroquinolone accumulation in bacteria and the effect of active efflux were measured by a fluorescence method. The outer membrane proteins of the bacteria were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). E. coli strains in this study included control strains JF701 and JF703 that are OmpC- or OmpF-deficient mutants of E. coli K-12, respectively, and the fluoroquinolone susceptible strain the fluoroquinolone susceptible strain of Escherichia coli (Ecs) and its in vitroselected resistant strains R2 and R256, and the clinical resistant isolates R5 and R6. Results The steady-state accumulation concentration of each drug in Ecs appeared to be the same as in JF701, while in the OmpF- deficient strain JF703, it was 1/5 CPLX or 1/2 LMLX lower than that in JF701, but JF703 was still susceptible to fluoroquinolones. On the other hand, compared with susceptible strains, a 2- to 10-fold decrease in the accumulation of each drug was found in the resistant strains except R2, in which the accumulation was slightly higher than in JF703. After the addition of 2,4-dinitrophenol (DNP), accumulation of each drug increased, especially in resistant strains, indicating that the function of the active efflux (pump) system in these bacteria had been enhanced dramatically. Furthermore, both OmpF and OmpC in Ecs, OmpF-deficiency in R2 and R256 and OmpC-deficiency in R5 and R6 were observed.Conclusion The decreased accumulation of hydrophilic fluoroquinolones in E. coli involved OmpF-deficiency and active efflux (pump), and the latter may be an important factor.

Effect of ozone on the performance of a hybrid ceramic membrane-biological activated carbon process

Two hybrid processes including ozonation-ceramic membrane-biological activated carbon （BAC） （Process A） and ceramic membrane-BAC （Process B） were compared to treat polluted raw water. The performance of hybrid processes was evaluated with the removal efficiencies of turbidity, ammonia and organic matter. The results indicated that more than 99% of particle count was removed by both hybrid processes and ozonation had no significant effect on its removal. BAC filtration greatly improved the removal of ammonia. Increasing the dissolved oxygen to 30.0 mg/L could lead to a removal of ammonia with concentrations as high as 7.80 mg/L and 8.69 mg/L for Processes A and B, respectively. The average removal efficiencies of total organic carbon and ultraviolet absorbance at 254 nm （UV254, a parameter indicating organic matter with aromatic structure） were 49% and 52% for Process A, 51% and 48% for Process B, respectively. Some organic matter was oxidized by ozone and this resulted in reduced membrane fouling and increased membrane flux by 25%-30%. However, pre-ozonation altered the components of the raw water and affected the microorganisms in the BAC, which may impact the removals of organic matter and nitrite negatively.

Shifts in microbial community structure and diversity in a MBR combined with worm reactors treating synthetic wastewater

The chemical oxygen demand（COD） and NH3-N removal, membrane fouling, sludge characteristics and microbial community structure in a membrane bioreactor（MBR） coupled with worm reactors（SSBWR） were evaluated for 210 days. The obtained results were compared to those from a conventional MBR（C-MBR） operated in parallel. The results indicated that the combined MBR（S-MBR） achieved higher COD and NH3-N removal efficiency,slower increase in membrane fouling, better sludge settleability and higher activities of the related enzymes in the activated sludge. Denaturing gradient gel electrophoresis was used to analyze the microbial community structures in the C-MBR and the S-MBR. The microbial community structure in the S-MBR was more diverse than that in the C-MBR. Additionally, the slow-growing microbes such as Saprospiraceae, Actinomyces, Frankia, Clostridium, Comamonas,Pseudomonas, Dechloromonas and Flavobacterium were enriched in the S-MBR, further accounting for the sludge reduction, membrane fouling alleviation and wastewater treatment.

A Facile Method for Dye and Heavy Metal Elimination by p H Sensitive Acid Activated Montmorillonite/Polyethersulfone Nanocomposite Membrane

Modified clay/polyethersulfone（PES） mixed matrix membranes（MMMs） were prepared by acid activated montmorillonite（AA-MMT） with different concentrations and used to eliminate dyes and remove heavy metals from aqueous solution. The morphology and physiochemical properties of prepared clay nanoparticles and MMMs were characterized using X-ray diffraction（XRD）, Fourier transform infrared（FTIR） spectroscopy, scanning electron microscopy（SEM）, enegy dispersive X-ray（EDX） spectroscopy, Brunauer-Emmett-Teller（BET） analysis, atomic force microscopy（AFM）, contact angle measurement and fouling studies. The filtration study showed that removal of dyes and heavy metals was strongly dependent on p H so that dyes with positive and negative charges showed different separation efficiency in acidic and alkaline conditions. The modified membranes possessed better heavy metal removal in acidic and alkaline p Hs. When the rejection of heavy metals was measured in an alkaline environment, it was observed that the rejection had a great increase compared to the neutral values for Zn^（2＋） and Ni^（2＋） ions, while rejection of Cu^（2＋） and Cd^（2＋） did not undergo significant changes. So it can be concluded that modified membranes show good selectivity for elimination of Zn^（2＋） and Ni^（2＋） ions with respect to other cations.

Research on Gliding Aerodynamic Effect of Deformable Membrane Wing for a Robotic Flying Squirrel

Inspired by creatures with membrane to obtain ultra-high gliding ability, this paper presents a robotic flying squirrel （a novel gliding robot） characterized as membrane wing and active membrane deformation. For deep understanding of membrane wing and gliding mechanism from a robotic system perspective, a simplified blocking aerodynamic model of the deformable membrane wing and CFD simulation are finished. In addition, a physical prototype is developed and wind tunnel experiments are carried out. The results show that the proposed membrane wing is able to support the gliding action of the robot. Meanwhile, factors including geometry characteristics, material property and wind speed are considered in the experiments to investigate the aerodynamic effects of the deformable membrane wing deeply. As a typical characteristic of robotic flying squirrel, deformation modes of the membrane wing not only affect the gliding ability, but also directly determine the effects of the posture adjustment. Moreover, different deformation modes of membrane wing are illustrated to explore the possible effects of active membrane deformation on the gliding performance. The results indicate that the deformation modes have a significant impact on posture adjustment, which reinforces the rationality of flying squirrel＇s gliding strategy and provides valuable information on prototype optimal design and control strategy in the actual gliding process.

Electrochemiluminescence Imaging of Liposome Permeabilization by an Antimicrobial Peptide:Melittin

The permeabilization of liposomes by melittin,an antimicrobial peptide(AMP),has been studied by an electrochemiluminescence(ECL)imaging strategy.The methodology consisted ffrst of encapsulating ECL reagents in sealed giant asymmetrical liposomes(100μm in diameter)made of DOPG/DOPC phospholipids(i.e.,1,2-dioleoyl-sn-glycerol-3-phospho-(1′-rac-glycerol)sodium salt/1,2-dioleolyl-sn-glycero-3-phosphocholine).Then liposomes were placed on an indium tin oxide electrode coated with poly-L-lysine to avoid any membrane poration/permeabilization through polarization of the electrode surface.Finally,the addition of melittin(from 10μM to 100 nM in concentration)enabled the permeabilization of the lipid membrane followed by the liposome content release and subsequent light generation through the ECL reagents oxidation processes.Interestingly,at a melittin concentration of 10μM,two successive leakages occurring on the same liposome could be imaged.Combination of ECL and photoluminescence imaging allowed comprehensive monitoring of the permeabilization and content release of a single liposome.This ECL imaging approach opens interesting perspectives to characterize the instant release of vesicle content upon permeabilization by AMPs or other membrane-active species.

Fabrication of self-assembling nanofibers with optimal cell uptake and therapeutic delivery efficacy

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy.In this contribution,we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides(MDPs)and use them as a model system to investigate structure-dependent cell penetrating activity.MDPs selfassembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane.However,despite the multivalent charge presentation,not all fibers led to high levels of membrane activity and cellular uptake.The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation.Nanofibers were found to sacrifice their dimension,thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction.The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation.In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone.Through these detailed structure and activity studies,the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.

Activation in the initial stage of oxygen permeation in SrCo_(0.9)Ta_(0.1)O_(3-δ)

Mixed conducting perovskite oxide SrCo_（0.9）Ta_（0.1）O_（3-δ）（SCT） is synthesized by solid-state reaction method.The activation in the initial stage of oxygen permeation through the SCT membrane is investigated.The results show that the activation in the initial stage of oxygen permeation has activate-memory,the first activation can only help to reduce active time of the next cycles,but it is helpless to increase the final oxygen permeation flux.XRD characterization shows that the imperfect perovskite phase structure is gradually improved and the crystal lattice has made some self-adjustment under the permeation conditions,therefore,the oxygen permeation flux of SCT disk membrane increases gradually and till it reaches a steady state.

The effect of tailing lipidation on the bioactivity of antimicrobial peptides and their aggregation tendency Special Issue:Emerging Investigators

Antimicrobial peptides(AMPs)are potentially powerful alternatives to conven-tional antibiotics in combating multidrug resistance,given their broad spectrum of activity.They mainly interact with cell membranes through surface electrostatic potentials and the formation of secondary structures,resulting in permeability and destruction of target microorganism membranes.Our earlier work showed that two leading AMPs,MSI-78(4–20)and pardaxin(1–22),had potent antimicrobial activ-ity against a range of bacteria.It is known that the attachment of moderate-length lipid carbon chains to cationic peptides can further improve the functionality of these peptides through enhanced interactions with the membrane lipid bilayer,inducing membrane curvature,destabilization,and potential leakage.Thus,in this work,we aimed to investigate the antimicrobial activity,oligomerization propensity,and lipid-membrane binding interactions of a range of N-terminal lipidated analogs of MSI-78(4–20)and pardaxin(1–22).Molecular modeling results suggest that aggregation of the N-lipidated AMPs may impart greater structural stability to the peptides in solu-tion and a greater depth of lipid bilayer insertion for the N-lipidated AMPs over the parental peptide.Our experimental and computationalfindings provide insights into how N-terminal lipidation of AMPs may alter their conformations,with subsequent effects on their functional properties in regard to their self-aggregation behavior,membrane interactions,and antimicrobial activity.