Taming membranes: functional immobilization of biological membranes in hydrogels

Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.     

Improvement of chloroperoxidase stability by covalent immobilization on chitosan membranes

Chloroperoxidase (CPO) from Caldariomyces fumago was optimally covalently immobilized on chitosan membranes pretreated with 0.8 M glutaraldehyde at pH 3.5 to give 3.18 mg CPO g⁻¹ support. Using monochlorodimedone (MCD) as assay substrate, the immobilized-CPO retained 40% activity at 50°C after 40 min whereas free CPO retained only 0.02%. The residual activity for immobilized-CPO was 99 and 58% compared with 68 and 43% for free CPO in the presence of 1.5 M urea and 300 μM H₂O₂, respectively, after 20 h.     

Finite element analysis of aortic root dilation: a new procedure to reproduce pathology based on experimental data

Sinotubular junction dilation is one of the most frequent pathologies associated with aortic root incompetence. Hence, we create a finite element model considering the whole root geometry; then, starting from healthy valve models and referring to measures of pathological valves reported in the literature, we reproduce the pathology of the aortic root by imposing appropriate boundary conditions. After evaluating the virtual pathological process, we are able to correlate dimensions of non-functional valves with dimensions of competent valves. Such a relation could be helpful in recreating a competent aortic root and, in particular, it could provide useful information in advance in aortic valve sparing surgery.     

Finite element analysis of aortic root dilation: a new procedure to reproduce pathology based on experimental data

Sinotubular junction dilation is one of the most frequent pathologies associated with aortic root incompetence. Hence, we create a finite element model considering the whole root geometry; then, starting from healthy valve models and referring to measures of pathological valves reported in the literature, we reproduce the pathology of the aortic root by imposing appropriate boundary conditions. After evaluating the virtual pathological process, we are able to correlate dimensions of non-functional valves with dimensions of competent valves. Such a relation could be helpful in recreating a competent aortic root and, in particular, it could provide useful information in advance in aortic valve sparing surgery.     

Effects of trypsin and cations on chloroplast membranes

A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl₂) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl₂) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the “diffuse electrical layer” hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism.     

Influence of antichaotropic electrolytes on the ferricyanide reduction of two immobilized systems: yeast-l-lactate dehydrogenase and chloroplast membranes

Summary The interactions between chloroplast membranes or yeast-l-lactate dehydrogenase and the microenvironment within artificial proteinaceous matrix were investigated. The activities of both immobilized systems were followed by the ferricyanide reduction. The influence of high ionic strength media (0.75 M potassium and sodium citrate, 1.0 M sodium sulphate) was studied. The results show in both immobilized systems an increase of the apparent activity and a better transformation of ferricyanide in the presence of high salt concentrations. Such behaviour can be explained by the high ionic strength of the external media, diffusional constraints and ionic exchange properties of the artificial matrix. The different mobilities of the anions and cations inside the matrix are believed to account for the observed variations in initial activities and stabilities. In particular we demonstrate that citrate and sulphate facilitate the penetration of the ferricyanide to the active reaction centres of the thylakoids and protect them against photoinactivation and denaturation.     

Bilayer lipid membranes supported on Teflon filters: a functional environment for ion channels

Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100-300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter=1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8±0.2 nF and membrane resistances of 25.9±4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1-80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1-10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.     

Influence of dolichyl phosphate on permeability and stability of bilayer lipid membranes

The ionic permeability coefficients, ionic transference numbers, activation energy of ion transport and breakdown voltage of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl 12-phosphate have been studied. The electrical measurements showed that dolichyl phosphate in phospholipid bilayers decreases membrane permeability, changes membrane ionic selectivity and increases membrane stability. These results are discussed in light of the aggregation behavior and the intramolecular clustering of a dolichyl phosphate molecule in phospholipid membranes. From our data we suggest that the hydrophilic part of dolichyl phosphate molecules regulates their behavior in membranes.     

Prevalence of mutagens in the environment: experimental data versus simulations

The recent availability of experimental data on the mutagenicity in Salmonella of a subset of high production volume chemicals allowed a comparison with the estimate derived from computer-based simulations. The prevalence of mutagens in the subsets was not significantly different: 20% versus 19.5% based upon experimental and simulation data, respectively. This provides support for the use of the rapid and low-cost simulation approach.     

Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3_chlM, molecular mass, 46402) and the portion of this factor homologous to bacterial IF-3 (IF-3_chlH, molecular mass 22829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3_chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3_chlM and IF-3_chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3_chlM and IF-3_chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3_chlH has at least 5-fold more activity than either native IF-3_chl or IF-3_chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3_chl lying outside of the homology domain may down-regulate the activity of this factor.This work was supported in part by National Institutes of Health Grant GM24963.     

Effect of the number of nucleic acid oligomer charges on the salt dependence of stability (DeltaG 37degrees) and melting temperature (Tm): NLPB analysis of experimental data

For nucleic acid oligomers with variable chain lengths, the salt concentration ([salt]) dependences of the denaturation temperature (T(m)) and of the free energy of helix formation at 37 degrees C (Delta) are predicted using nonlinear Poisson-Boltzmann (NLPB) calculations. Analysis of experimental data reveals that the ratio of the [salt] derivative of melting temperature (ST(m) = dT(m)/d log[salt]) to the value for a polymer with the same base composition (ST(m)/ST(m, infinity)) is independent of base composition but strongly dependent on the number of DNA charges (/Z/) below approximately 8 bp for two-strand helices (formed from association of two complementary strands) and below approximately 18 bp for hairpin helices (formed from folding of one self-complementary strand). We interpret these ST(m)/ST(m, infinity) ratios in terms of the ratio of thermodynamic ion release from the oligomer (Deltan(u), per charge) to that from the same oligomer embedded in polymeric DNA (Deltan(u, infinity), per charge). Experimental values of ST(m)/ST(m, infinity) and its dependence on /Z/ are in good agreement with NLPB predictions for a preaveraged (essential structural) model of DNA. In particular, the NLPB calculations describe the stronger /Z/ dependence of ST(m) observed for melting of oligomeric hairpin helices than for melting of two-strand helices. These calculations predict an experimentally detectable (>or=10%) difference between ST(m) and ST(m, infinity) which increases strongly with decreasing length for two-strand helix lengths of <15 bp and for hairpin helix lengths of <30 bp. From NLPB values of Deltan(u)/Deltan(u, infinity), we predict Delta as a function of [salt] and /Z/. Predictions of thermodynamic and thermal stabilities of oligomeric helices as functions of length and [salt] are consistent with and represent a significant refinement of the average oligomer salt effect currently in use in nearest neighbor stability predictions.     

Effects of alpha-tocopherol (vitamin E) on the stability and lipid dynamics of model membranes mimicking the lipid composition of plant chloroplast membranes

Tocopherol (vitamin E) is widely recognized as a cellular antioxidant. It is essential for human and animal health, but only synthesized in photosynthetic organisms, where it is localized in chloroplast membranes. While many studies have investigated non-antioxidative effects of tocopherol on phospholipid membranes, nothing is known about its effects on membranes containing chloroplast glycolipids. Here, liposomes resembling plant chloroplast membranes were used to investigate the effects of alpha-tocopherol on vesicle stability during freezing and on lipid dynamics. alpha-Tocopherol had a pronounced influence on membrane dynamics and showed strong interactions in its effects on membrane stability during freezing with the cryoprotectant sucrose. alpha-Tocopherol showed maximal effects at low concentrations (around 2mol%), close to its contents in chloroplast membranes.     

Economic growth and its impact on environment: A panel data analysis

This paper aims to explore the relationship between the economic growth and the pressure on nature from the environmental sustainability perspective. The pressure on nature is measured by the natural disinvestment component of the Adjusted Net Savings data of the World Bank; which is the sum of energy, mineral, net forest depletions and carbon dioxide damage, all measured in US dollars. Our panel consists of 213 countries and spans the period between 1970 and 2008. We employ a panel Fixed-Effects Instrumental Variable (IV) methodology. Regression analysis reveals that there is a positive relationship between income and pressure on nature, yet the effect is much stronger in middle-income than in low and high-income countries, and also differs across subcomponents. While increase in income decreases the pressure on forests, CO₂ damage and mineral extraction increase. We found that increasing trade, all else equal, increases the pressure on nature, hence our findings support Race-to-the-bottom hypothesis.     

不同水盐梯度下功能多样性和功能冗余对荒漠植物群落稳定性的影响

功能多样性和功能冗余是物种多样性的两个组成部分,也是影响群落稳定性的两个重要因素。基于不同水盐梯度下植物功能多样性、功能冗余、物种多样性和群落稳定性及其相关关系的计算结果,分析功能多样性和功能冗余对群落稳定性的影响,结果表明:(1)功能多样性、物种多样性和群落稳定性均表现为高水高盐和中水中盐群落显著高于低水低盐群落(P0.05);(2)高水高盐群落,功能多样性与物种多样性的相关系数小于功能冗余与物种多样性的相关系数,且功能多样性与稳定性的相关系数也小于功能冗余与稳定性的相关系数,而中水中盐和低水低盐群落的相关系数则呈现相反的规律;(3)中水中盐和低水低盐群落的功能多样性的标准化偏回归系数均大于功能冗余的标准化偏回归系数;(4)典范对应分析中,土壤含水量可以解释总特征根的22.7%,而土壤含盐量仅可以解释总特征根的1.3%;(5)高水高盐群落的稳定比最接近20/80,稳定性最高;低水低盐群落远离20/80,稳定性最低。改进后的Godron稳定性测定方法与物种种群密度变异系数方法得出的结果相同。综上可知,功能多样性和功能冗余两者中与物种多样性关系更为密切者对群落稳定性的影响也越大,且两者均可提高群落稳定性,也就证明冗余假说在温带干旱荒漠区域的隐域性植物群落中是成立的;群落稳定性、功能多样性、功能冗余及物种多样性主要是受土壤含水量的影响,土壤含盐量对其影响较小。     

Prostaglandin-H-synthase: stability and activity during immobilization on silica gel]

Prostaglandin H synthase was isolated in the form of microsomes from sheep vesicular glands and immobilized on silica gel. This system of prostaglandin synthesis was activated by calcium ions and stabilized by adrenaline. Microsomes immobilized in the presence of adrenaline and calcium ions were stable upon storage at 4 degrees C. After two months, their activity was 80% of the initial activity. Immobilized microsomes were able to catalyze several cycles of prostaglandin E2 synthesis with no substantial loss of activity: after eight utilization cycles, they retained 66% of their initial enzymic activity.     

Combined phylogenetic analysis in the Rubiaceae-Ixoroideae: morphology, nuclear and chloroplast DNA data

Parsimony analyses of morphology, restriction sites of the cpDNA, sequences from the nuclear, ribosomal internal transcribed spacer (ITS), and the chloroplast gene rbcL were performed to asses tribal and generic relationships in the subfamily Ixoroideae (Rubiaceae). The tribes Vanguerieae and Alberteae (Antirheoideae) are clearly part of Ixoroideae, as are some Cinchonoideae taxa. Pavetteae should exclude Ixora and allies, which should be recognized as the tribe Ixoreae. Heinsenia, representing Aulacocalyceae, is part of Gardenieae, as is Duperrea, a genus earlier placed in Pavetteae. Posoqueria and Bertiera and the taxa in the subtribe Diplosporinae should be excluded from Gardenieae. Bertiera and three Diplosporinae taxa are part of Coffeeae, while Cremaspora (Diplosporinae) is best housed in a tribe of its own, Cremasporeae. The mangrove genus Scyphiphora, recently placed in Diplosporinae, is closer to Ixoreae and tentatively included there. The combined analysis resulted in higher resolution compared to the separate analyses, exemplifying that combined analyses can remedy the incapability of one data set to resolve portions of a phylogeny. Twenty-four new rbcL sequences representing all five Ixoroideae tribes (sensu Robbrecht) are presented.     

Lipase-catalyzed solid-phase synthesis of sugar esters. Influence of immobilization on productivity and stability of the enzyme

The lipase-catalyzed synthesis of 6-O-glucose palmitate in a mainly solid-phase system was investigated. Lipase from Candida antarctica B was immobilized on various carriers by physical absorption or covalent binding. Highest conversion (84%, 24 h) and productivity (0.69 mmol product per gram lipase and hour) were achieved with lipase immobilized on polypropylene (EP 100), whereas other carriers gave at maximum 46% conversion. A good agreement between aquaphilicity of the carrier and conversion was found. The lipase was reused six times and conversion decreased by only approximately 25%. The influence of temperature, organic solvent and fatty acid chain length on lipase stability was also investigated. For the latter, a correlation between the log P of the fatty acid (determined from hydrophobic fragment constants) and lipase stability was observed. Palmitic acid and the corresponding vinyl ester gave highest conversion in the acylation of β-d(+)-glucose, whereas tripalmitin, palmitic acid anhydride and the methyl ester allowed only low conversion. Beside β-d(+)-glucose, also other monosaccharides and trehalose were acylated.