Anionic CO2 activation in the anionic and di-anionic state of aza-naphthalene

Nitrogen-containing polycyclic aromatic hydrocarbon (PAH) is the single basic moiety in N-doped graphene, the only metal-free catalyst reported to date to successfully produce the oxygen reduction reaction. N-doped graphene is quite promising as a material to increase the efficiency of oxygen reduction. In addition, it is known that when carbon dioxide is added to aza-benzene, there will be an associative chemical reaction upon electron attachment between the anionic nitrogen atoms in the aza-benzene and the carbon atom in the carbon dioxide; however, it has previously been reported that when there are more nitrogen atoms in the small aza-benzene moiety, the associative reaction does not always occur. In this study, we carried out a theoretical simulation to determine whether more electrons increase the CO₂ reductive reactivity of the aza-naphthalene as a model system of a nitrogen-containing polycyclic aromatic hydrocarbon. We found that even though an associative chemical reaction between nitrogen atoms in the N-PAH and carbon atoms in carbon dioxide did not occur in anionic complexes of aza-naphthalene and carbon dioxide, chemical reactions did occur in all the nitrogen atoms of these complexes when we added an extra excess electron. Therefore, we conclude that the efficiency of CO₂ reduction will be increased in nitrogen atoms when more electrons are added to increase their anionic properties.

Nitrogen-containing polycyclic aromatic hydrocarbon (PAH) is the single basic moiety in N-doped graphene, the only metal-free catalyst reported to date to successfully produce the oxygen reduction reaction.     

Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidine-rich peptides at acidic pH

The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state (2)H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.     

Dehydroabietic acid, a major anionic contaminant of pulp mill effluent, reduces both active p-aminohippurate transport and passive membrane permeability in isolated renal membranes

The renal organic anion transport system plays a pivotal role in elimination of potentially toxic anions. This system is driven by indirect coupling to the sodium gradient at the basolateral membrane, i.e., the organic anion enters the cell in exchange for internal alpha-ketoglutarate (alpha KG) and the in greater than out alpha KG gradient is regenerated by Na+/alpha KG cotransport. The resin acid, dehydroabietic acid (DHAA), is one of several anionic xenobiotics which enter the environment secondary to pulp and paper processing. Because it is largely ionized at neutral pH (pKa, 5.7), DHAA should share the organic anion system. Indeed, Na+/glutarate-coupled p-aminohippurate (PAH) uptake by renal basolateral membrane vesicles was inhibited competitively by DHAA (Ki congruent to 150 microM). Despite the reduced rate of PAH uptake, a substantial, but delayed, overshoot was observed, suggesting additional effects. Passive permeabilities to mannitol, PAH and sodium were all decreased by DHAA, consistent with a general tightening of the membrane. Decreased permeability extended the effective lifetime of imposed ion gradients. Thus, sodium driven glutarate uptake was stimulated by 200 microM DHAA, prolonging and more than doubling its overshoot. Because the immediate driving force for PAH uptake into basolateral membrane vesicles is the magnitude of the glutarate gradient, DHAA increased the driving force for PAH uptake and permitted a substantial overshoot despite the reduced rate of PAH uptake. These data indicate that DHAA has several distinctly different effects on the membrane.     

Salvinorin A: allosteric interactions at the mu-opioid receptor

Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic kappa-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [(3)H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E(MAX) values of 78.6, 72.1, and 45.7%, respectively, and EC(50) values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [(3)H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E(MAX) values of 86.2, 64, and 33.6%, respectively, and EC(50) values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [(3)H]DAMGO showed that Salvinorin A (10 and 30 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Saturation binding studies with [(3)H]diprenorphine showed that Salvinorin A (10 and 40 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [(3)H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [(3)H]DAMGO and [(3)H]diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.     

Lipid membrane interactions of self-assembling antimicrobial nanofibers: effect of PEGylation

Supramolecular assembly and PEGylation (attachment of a polyethylene glycol polymer chain) of peptides can be an effective strategy to develop antimicrobial peptides with increased stability, antimicrobial efficacy and hemocompatibility. However, how the self-assembly properties and PEGylation affect their lipid membrane interaction is still an unanswered question. In this work, we use state-of-the-art small angle X-ray and neutron scattering (SAXS/SANS) together with neutron reflectometry (NR) to study the membrane interaction of a series of multidomain peptides, with and without PEGylation, known to self-assemble into nanofibers. Our approach allows us to study both how the structure of the peptide and the membrane are affected by the peptide–lipid interactions. When comparing self-assembled peptides with monomeric peptides that are not able to undergo assembly due to shorter chain length, we found that the nanofibers interact more strongly with the membrane. They were found to insert into the core of the membrane as well as to absorb as intact fibres on the surface. Based on the presented results, PEGylation of the multidomain peptides leads to a slight net decrease in the membrane interaction, while the distribution of the peptide at the interface is similar to the non-PEGylated peptides. Based on the structural information, we showed that nanofibers were partially disrupted upon interaction with phospholipid membranes. This is in contrast with the considerable physical stability of the peptide in solution, which is desirable for an extended in vivo circulation time.

Wrane interaction of a series of self-assembling antimicrobial peptides with and without PEGylation using small angle X-ray and neutron scattering and neutron reflectometry.     

Sonoluminescence as an indicator of cell membrane disruption by acoustic cavitation

Ultrasound (US) has been shown to transiently disrupt cell membranes and, thereby, facilitate the loading of drugs and genes into viable cells. Because these effects are believed to be mediated by cavitation, we hypothesized that measured levels of cavitation-induced sonoluminescence should correlate with levels of US bioeffects. We, therefore, quantified the number of calcein molecules delivered and the loss of viability in prostate cancer cells exposed to 24-kHz US over a range of different pulse lengths (1 to 100 ms), total exposure times (0.1 to 10 s) and pressures (1.0 to 9.8 atm). Consistent with previous observations, uptake increased and viability decreased with increasing pulse length, total exposure time and pressure. As a new observation, we established correlations between the amount of light produced by sonoluminescence and both molecular uptake and cell viability. These results support a cavitation-based mechanism for these bioeffects and suggest a means to control US effects on cells using sonoluminescence-based feedback.     

The design and synthesis of polymers for eukaryotic membrane disruption.

The intracellular trafficking of drugs is critical to the efficacy of drugs that are susceptible to attack by lysosomal enzymes. It is therefore an important goal to design and synthesize molecules which can enhance the transport of endocytosed drugs from the endosomal compartments to the cytoplasm. The pH of an endosome is lower than that of the cytosol by one to two pH units, depending on the stage of endosomal development. This pH gradient is a key factor in the design of membrane-disruptive polymers which could enhance the endosomal release of drugs. Such polymers should disrupt lipid bilayer membranes at pH 6.5 and below, but should be non-lytic at pH 7.4. We have designed and synthesized pH-sensitive synthetic polymers which efficiently disrupt red blood cells within a sharply defined pH range. One of these polymers, poly(ethyl acrylic acid) (PEAAc) has been previously shown to disrupt synthetic vesicles in a pH-dependent fashion [6]. PEAAc hemolyzes red blood cells with an activity of 10(7) molecules per red blood cell, which is as efficient on a molar basis as the peptide melittin. The mechanism of RBC hemolysis by PEAAc is consistent with the colloid osmotic mechanism. PEAAc's hemolytic activity rises rapidly as the pH decreases from 6.3 to 5.0, and there is no hemolytic activity at pH 7.4. A related polymer, poly(propyl acrylic acid) (PPAAc), was synthesized to test whether making the pendant alkyl group more hydrophobic by adding one methylene group would increase the hemolytic activity. PPAAc was found to disrupt red blood cells 15 times more efficiently than PEAAc at pH 6.1. PPAAc was also not active at pH 7.4 and displayed a pH-dependent hemolysis that was shifted toward higher pH's. Random 1:1 copolymers of ethyl acrylate (EA) and acrylic acid (AAc) (which contain random -COOH and -C(2)H(5) groups that are present and regularly repeat in PEAAc) also displayed significant hemolytic activity, with an efficiency close to PEAAc. These results demonstrate that pH-sensitive synthetic polymers can be molecularly engineered to efficiently disrupt eukaryotic membranes within defined and narrow pH ranges. Thus, these polymers might serve as endosomal disruptive agents with specificities for early or late endosomes.     

Protein–membrane interactions: blood clotting on nanoscale bilayers

Summary.  The clotting cascade requires the assembly of protease–cofactor complexes on membranes with exposed anionic phospholipids. Despite their importance, protein–membrane interactions in clotting remain relatively poorly understood. Calcium ions are known to induce anionic phospholipids to cluster, and we propose that clotting proteins assemble preferentially on such anionic lipid-rich microdomains. Until recently, there was no way to control the partitioning of clotting proteins into or out of specific membrane microdomains, so experimenters only knew the average contributions of phospholipids to blood clotting. The development of nanoscale membrane bilayers (Nanodiscs) has now allowed us to probe, with nanometer resolution, how local variations in phospholipid composition regulate the activity of key protease–cofactor complexes in blood clotting. Furthermore, exciting new progress in solid-state NMR and large-scale molecular dynamics simulations allow structural insights into interactions between proteins and membrane surfaces with atomic resolution.     

Review article: platelet-collagen interactions: membrane receptors and intracellular signalling pathways

Platelet adhesion to and activation by exposed subendothelial collagen plays a critical role in normal haemostasis and pathological thrombosis. Recent advances in elucidating the mechanisms underlying platelet-collagen interaction support a 'two-site, two-step' model. Direct platelet binding to integrin alpha2beta1 mainly sustains adhesion and allows recognition of glycoprotein VI. The latter interaction is responsible for characteristic intracellular signalling events leading to p72Syk and PLCgamma2 activation. The present review describes the known collagen receptors on platelets and discusses the current understanding of signal transduction promoted by collagen.     

Quantification of C60-induced membrane disruption using a quartz crystal microbalance

Direct contact between fullerene C₆₀ nanoparticles (NPs) and cell membranes is one of mechanisms for its cytotoxicity. In this study, the influence of C₆₀ NPs on lipid membranes was investigated. Giant unilamellar vesicles (GUVs) were used as model cell membranes to observe the membrane disruption after C₆₀ exposure. C₆₀ NPs disrupted the positively charged GUVs but not the negatively charged vesicles, confirming the role of electrostatic forces. To quantify the C₆₀ adhesion on membrane and the induced membrane disruption, a supported lipid bilayer (SLB) and a layer of small unilamellar vesicles (SUVs) were used to cover the sensor of a quartz crystal microbalance (QCM). The mass change on the SLB (Δm_SLB) was caused by the C₆₀ adhesion on the membrane, while the mass change on the SUV layer (Δm_SUV) was the combined result of C₆₀ adhesion (mass increase) and SUV disruption (mass loss). The surface area of SLB (A_SLB) was much smaller than the surface area of SUV (A_SUV), but Δm_SLB was larger than Δm_SUV after C₆₀ deposition, indicating that C₆₀ NPs caused remarkable membrane disruption. Therefore a new method was built to quantify the degree of NP-induced membrane disruption using the values of Δm_SUV/Δm_SLB and A_SUV/A_SLB. In this way, C₆₀ can be compared with other types of NPs to know which one causes more serious membrane disruption. In addition, C₆₀ NPs caused negligible change in the membrane phase, indicating that membrane gelation was not the mechanism of cytotoxicity for C₆₀ NPs. This study provides important information to predict the environmental hazard presented by fullerene NPs and to evaluate the degree of membrane damage caused by different NPs.

Fullerene C₆₀ NPs adhere on lipid membrane due to electrostatic force and cause membrane disruption.     

Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid

Pyrazinamide is an important sterilizing drug that shortens tuberculosis (TB) therapy. However, the mechanism of action of pyrazinamide is poorly understood because of its unusual properties. Here we show that pyrazinoic acid, the active moiety of pyrazinamide, disrupted membrane energetics and inhibited membrane transport function in Mycobacterium tuberculosis. The preferential activity of pyrazinamide against old non-replicating bacilli correlated with their low membrane potential and the disruption of membrane potential by pyrazinoic acid and acid pH. Inhibitors of membrane energetics increased the antituberculous activity of pyrazinamide. These findings shed new light on the mode of action of pyrazinamide and may help in the design of new drugs that shorten therapy.     

Synergy between mu opioid ligands: evidence for functional interactions among mu opioid receptor subtypes

Pharmacological differences among mu opioid drugs have been observed in in vitro and in vivo preclinical models, as well as clinically, implying that all mu opioids may not be working through the same mechanism of action. Here we demonstrate analgesic synergy between L-methadone and several mu opioid ligands. Of the compounds examined, L-methadone selectively synergizes with morphine, morphine-6beta-glucuronide, codeine, and the active metabolite of heroin, 6-acetylmorphine. Morphine synergizes only with L-methadone. In analgesic assays, D-methadone was inactive alone and did not enhance morphine analgesia when the two were given together, confirming that L-methadone was not acting through N-methyl-D-aspartate mechanisms. Both L-methadone and morphine displayed only additive effects when paired with oxymorphone, oxycodone, fentanyl, alfentanyl, or meperidine. Although it displays synergy in analgesic assays, the L-methadone/morphine combination does not exhibit synergy in the gastrointestinal transit assay. This analgesic synergy of L-methadone with selective mu opioid drugs and the differences in opioid-mediated actions suggest that these drugs may be acting via different mechanisms. These findings provide further evidence for the complexity of the pharmacology of mu opioids.     

Screening of different interactions in oxo-manganese porphyrin dimers containing axial N-donor ligands: a theoretical study

A theoretical analysis for describing the dimeric assemblies of high-valent manganese(v)-oxo meso-tetraphenylporphyrin (TPP) ([(TPP)Mn^VO]₂²⁺) and meso-tetrakis(pentafluorophenyl)porphyrin (TPFPP) ([(TPFPP)Mn^VO]₂²⁺) in the presence of axial N-donor ligands is presented. Our theoretical results revealed two types interactions in dimers: a sandwich-like interaction between phenyl rings of porphyrin molecules, and a non-bonded T-shape interaction between nitrogen donors attached to Mn centers. The curvature in the geometry of porphyrin in the [(TPP)Mn^VO]₂²⁺/N-donor system is significantly smaller than that of [(TPFPP)Mn^VO]₂²⁺/N-donor system. Moreover, the Mn–N_(ax) distances in [(TPFPP)Mn^VO]₂²⁺/N-donor system are shorter than those of [(TPP)Mn^VO]₂²⁺/N-donor system. Also, the donor–acceptor interaction between the imidazoles and the Mn centers are stronger than those of the other ligands in both porphyrins. These results are supported by atoms in molecules (AIM) and natural bond orbital (NBO) analysis.

A DFT analysis for describing the dimeric assemblies of high-valent manganese(v)-oxo meso-tetraphenylporphyrin ([(TPP)Mn^VO]₂²⁺) and meso-tetrakis(pentafluorophenyl)porphyrin ([(TPFPP)Mn^VO]₂²⁺) in the presence of axial N-donor ligands is presented.     

Selective membrane disruption: mode of action of C16G2, a specifically targeted antimicrobial peptide

The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.     

Ketolide antimicrobial activity persists after disruption of interactions with domain II of 23S rRNA

Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity. All macrolides and ketolides bind to the 50S ribosomal subunit, where they come into contact with adenosine 2058 (A2058) within domain V of the 23S rRNA and block protein synthesis. An additional interaction at nucleotide A752 in the rRNA domain II is made via the synthetic carbamate-alkyl-aryl substituent in the ketolides HMR3647 (telithromycin) and HMR3004, and this interaction contributes to their improved activities. Only a few macrolides, including tylosin, come into contact with domain II of the rRNA and do so via interactions with nucleotides G748 and A752. We have disrupted these macrolide-ketolide interaction sites in the rRNA to assess their relative importance for binding. Base substitutions at A752 were shown to confer low levels of resistance to telithromycin but not to HMR3004, while deletion of A752 confers low levels of resistance to both ketolides. Mutations at position 748 confer no resistance. Substitution of guanine at A2058 gives rise to the MLS(B) (macrolide, lincosamide, and streptogramin B) phenotype, which confers resistance to all the drugs. However, resistance to ketolides was abolished when the mutation at position 2058 was combined with a mutation in domain II of the same rRNA. In contrast, the same dual mutations in rRNAs conferred enhanced resistance to tylosin. Our results show that the domain II interactions of telithromycin and HMR3004 differ from each other and from those of tylosin. The data provide no indication that mutations within domain II, either alone or in combination with an A2058 mutation, can confer significant levels of telithromycin resistance.     

钛合金种植体表面制备的生物活性膜

背景:传统的仿生方法通常采用常规模拟体液和1.5倍模拟体液,其缺点就在于涂层生长过于缓慢,生长周期长达数周甚至几个月.目的:探讨3倍模拟体液诱导Ti-6Al-4V合金表面快速形成类羟基磷灰石涂层的能力.设计、时间及地点:体外生物学分析.实验于2008-11/2009-03在中国科学院金属研究所完成.材料:Ti-6Al-4V合金片由陕西省宝鸡市博达金属材料有限公司提供.方法:将经过预处理的Ti-6Al-4V合金浸泡在37℃的3倍模拟体液中,为保持浸泡液成分的恒定,溶液每24 h更换1次,共浸泡3 d.主要观察指标:用扫描电镜、X射线衍射仪和红外光谱仪分析涂层的形貌、相及元素组成.结果:扫描电镜观察样品浸泡1 d时,表面被沉积膜覆盖,局部区域有较大颗粒状的晶体出现.浸泡3 d时,表面完全被沉积膜覆盖,且有大量颗粒状或鳞片状晶体出现,晶体沉积以某一区域为核心,持续生长.生成物主要由Ca,P,O,C等元素组成,但是没有检测到Al,V等元素的存在.X射线衍射仪和红外光谱仪分析浸泡3 d时Ti-6Al-4V合金表面制备的生物活性膜主要成分为羟基磷灰石.结论:使用3倍模拟体液可以快速诱导Ti-6Al-4V合金表面类羟基磷灰石的沉积,显著缩短涂层形成的时间.