Thermal induced modification of the contact mechanics of adhering liposomes on cationic substrate

The correlation between the mechanical property and the thermotropic transition of the phospholipid bilayer has been recently demonstrated (Chem. Phys. Lipids 110 (2001) 27). However, the role of thermal induced mechanical responses of phospholipid bilayer on the contact mechanics of liposome adhering on a cationic substrate has not been determined. In this study, confocal-reflectance interference contrast microscopy, phase contrast microscopy and contact mechanics modeling are applied to probe the adhesion mechanisms of liposomes in the presence of electrostatic interactions during the thermotropic transition of the lipid bilayer. When temperature increases from 23 to 49 °C at pH 7.4, the degree of liposome deformation (a/R) and adhesion energy of dipalmitoyl-sn-glycero-3-phosphocholine liposome increases by 10% and remains constant, respectively, on 3-amino-propyl-triethoxy-silane (APTES) modified substrate. The extents of increase in these two parameters are highly dependent on the physicochemical properties of the rigid substrate. At pH 4, the adhesion energies above and below the phase transition temperature (T_m) are increased by one order of magnitude due to the formation of the free silanol groups on APTES substrate. In hypotonic condition, the degree of vesicle deformation remains constant and the adhesion energy reduces by 20% during sample heating. Under all conditions, the adhesion energy of the adhering liposome spans a few orders of magnitude against the increase of liposome size as the surface area to volume ratio is maximized in smallest vesicle.     

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L-MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N-terminal end of the L-MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L-MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.     

If space is provided,bulky modification on the rim of azurin's beta-barrel results in folded protein

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.     

Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

活性X-3B红染料在水-土壤-作物连续体中的迁移模型研究

进入环境的自学成才性X-3B红染料是一典型的持久性有机污染物（POPs)。它从水分室向土壤分室再到作物分室的迁移,是一连续的生态过程,基于此,从理论上对活性X-3B红染料在水-土壤两相,土壤-作物两相以及水-土壤-作物整个连续体中的迁移进行了定量描述与探讨,并采用棕壤-大豆、褐土-小麦,红壤-萝卜和水稻土-水稻等系统给予了实验论证,表明活性X-3B红染料通过吸附机制从水分室到土壤分室的迁移符合Langmuir模型。通过作物根系吸收机制从土壤分室到作物分室的迁移可以用对数化的作物-土壤积累因子（CSAF）模型予以定量描述。     

Use of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase

Understanding protein aggregation may hold important clues to understanding what goes wrong with protein folding in neurodegenerative disorders and in bioreactors in which proteins are overexpressed. Unfortunately, aggregates tend to be intractable to most standard methods of biochemical investigation. Thus, relatively little is even now known about the micro- and macro-structural features of aggregates. To gain insights into the thermal aggregation of a model globular protein [bovine carbonic anhydrase (BCA)], we have used spectrofluorimetry to examine the binding of a hydrophobic dye, 8-anilinonaphthalene sulfonate (ANS), to hydrophobic clusters on the protein's surface both before and after heat-induced aggregation and upon cooling. Whereas native BCA shows no surface hydrophobicity, thermally aggregated BCA displays significant hydrophobicity both in the heated state and upon cooling. The timing of the addition of ANS in the course of aggregation makes no net difference to the ANS bound; we argue that this suggests that aggregates are essentially porous. Cooling of aggregates results in a dramatic, fully reversible increase in ANS binding that cannot be explained by the temperature dependence of fluorescence quantum yield alone; we argue that the enhancement of fluorescence upon cooling indicates possible structural consolidation of unfolded regions within aggregates (akin to refolding), with the required structural reorganization being facilitated by porosity. Finally, implications of porosity in aggregates are discussed, in particular, for the possible immobilization of enzymes through fusion with aggregation-prone protein domains.     

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry–Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, ΔG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed ΔG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E_a, as derived from the kinetic stability analysis.     

A bioresorbable, polylactide reservoir for diffusional and osmotically controlled drug delivery

The purpose of this study was to design and characterize a zero-order bioresorbable reservoir delivery system (BRDS) for diffusional or osmotically controlled delivery of model drugs including macromolecules. The BRDS was manufactured by casting hollow cylindrical poly (lactic acid) (PLA): polyethylene glycol (PEG) membranes (10 x 1.6 mm) on a stainless steel mold. Physical properties of the PLA:PEG membranes were characterized by solid-state thermal analysis. After filling with drug (5 fluorouracil [5FU] or fluorescein isothiocyanate [FITC]-dextran:mannitol, 5:95 wt/wt mixture) and sealing with viscous PLA solution, cumulative in vitro dissolution studies were performed and drug release monitored by ultraviolet (UV) or florescence spectroscopy. Statistical analysis was performed using Minitab (Version 12). Differential scanning calorimetry thermograms of PLA:PEG membranes dried at 25 degrees C lacked the crystallization exotherms, dual endothermal melting peaks, and endothermal glass transition observed in PLA membranes dried at -25 degrees C. In vitro release studies demonstrated zero-order release of 5FU for up to 6 weeks from BRDS manufactured with 50% wt/wt PEG (drying temperature, 25 degrees C). The release of FITC dextrans of molecular weights 4400, 42 000, 148 000, and 464 000 followed zero-order kinetics that were independent of the dextran molecular weight. When monitored under different concentrations of urea in the dissolution medium, the release rate of FITC dextran 42 000 showed a linear correlation with the calculated osmotic gradient(DeltaPi). This study concludes that PEG inclusion at 25 degrees C enables manufacture of uniform, cylindrical PLA membranes of controlled permeability. The absence of molecular weight effects and a linear dependence of FITC-dextran release rate on DeltaPi confirm that the BRDS can be modified to release model macromolecules by an osmotically controlled mechanism.     

The Use Of Methods For Injury Determination And Quantification From Natural Resource Damage Assessment In Ecological Risk Assessment

Several Federal statutes provide the government the authority to recover natural resource damages including the Clean Water Act Amendments (1977), the Outer Continental Shelf Act Amendments (1978), the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) of 1980, and the Oil Pollution Act of 1990 (OPA 90). CERCLA and OPA 90 are the principle Federal statutes which authorize trustees to assess damages for trust resources which are lost, injured, or destroyed as a result of the discharge of oil or the release of hazardous substances. The Department of the Interior was charged with developing natural resource damage assessment (NRDA) regulations and procedures from CERCLA and the Department of Commerce, National Oceanic and Atmospheric Administration (NOAA) was charged with developing regulations for OPA 90. NRDA is a process for making the public “whole” for direct injury to natural resources and/or the services of natural resources. The primary objectives of the NRDA process are to identify and quantify natural resource injury, determine the damages resulting from the injury, and develop and implement appropriate restoration actions. The goal is to be accomplished by implementing a plan for the restoration, rehabilitation, replacement, or acquisition of equivalent natural resources. NRDA is an after-the-fact process; however, the OPA 90 NRDA rules describe a pre-spill planning process. The models and formulae can be used in this pre-spill planning process to estimate damages (both ecological and economic). Some of those tools, in particular NRDAMCME, have uses in ranking risk from spills of petroleum products and hazardous substances. This information can be used to protect ecological resources and lower NRDA costs through pre spill planning and management of resources during a spill.     

Phenological mapping in a topographically complex landscape by combining field survey with an irradiation model

Abstract. A phenological map has been prepared for the Podyjí/Thayatal National Park, located on the border between the Czech Republic and Austria. The area is characterized by V-shaped river valleys deeply incised into a gently undulating landscape. Five stages of phenological development were defined, based upon phenological spectra of plant communities in late April; 96 sites, more or less regularly spaced over the area, were assigned to one of these stages using field observations of these spectra. Potential direct solar irradiation was calculated from a digital elevation model for a 25 m x 25 m grid extending over the study area. A method was developed for interpolation of phenological observations by weighted regression of phenology on the irradiation model. This interpolation enables the prediction of the local stage of phenological development across the study area without levelling out phenological patterns dependent upon small-scale topographic variation. Spring phenological events appear to be more advanced in the valleys (except for steep north-facing slopes) than in the adjacent landscape. Possible climatic processes underlying this pattern are discussed with emphasis on temperature inversions in the river valleys. Cold-air-drainage inversions reported from the valleys are probably too infrequent and of too short duration to delay plant phenology. The combined effects of advanced phenology and the increased risk of spring frost injury due to these inversions in the valleys may be an explanation for local distributional patterns of flora and vegetation.