Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers

This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air–water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 mN m⁻¹, the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.     

A Novel β-Glucosidase from Humicola insolens with High Potential for Untreated Waste Paper Conversion to Sugars

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116 kDa (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and 404 kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65 °C, respectively, and the enzyme was stable for 60 min at 50 °C, maintaining 71 % residual activity after 60 min at 55 °C. BglHi2 hydrolyzed p-nitrophenyl-β-d-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (K _M?=?0.24?±?0.01 mmol L^?1) and catalytic efficiency (k _cat/K _M?=?1,304.92?±?53.32 L mmol^?1 s^?1). The activity was insensitive to Fe⁺³, Cr⁺², Mn⁺², Co⁺², and Ni²⁺, and 50–60 % residual activities were retained in the presence of Pb²⁺, Hg²⁺, and Cu²⁺. Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5 L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24–48 h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100 % glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.     

Magnetoelectric effect in cobalt ferrite-barium titanate composites and their electrical properties

CoFe₂O₄-BaTiO₃ composites were prepared using conventional ceramic double sintering process with various compositions. Presence of two phases in the composites was confirmed using X-ray diffraction. The dc resistivity and thermoemf as a function of temperature in the temperature range 300 K to 600 K were measured. Variation of dielectric constant (ɛ′) with frequency in the range 100 Hz to 1 MHz and also with temperature at a fixed frequency of 1 kHz was studied. The ac conductivity was derived from dielectric constant (ɛ′) and loss tangent (tan δ). The nature of conduction is discussed on the basis of small polaron hopping model. The static value of magnetoelectric conversion factor has been studied as a function of magnetic field.     

Adsorption of detergent-solubilized and phospholipase C-solubilized alkaline phosphatase at air/liquid interfaces

To investigate the influence of a hydrophobic anchor on protein adsorption, equilibrium and dynamic aspects of the adsorption of two different solubilized forms of rat osseous plate alkaline phosphatase on Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) were studied. Surface pressure and surface potential measurements at air/liquid interfaces were carried out using the detergent-solubilized form (DSAP) of alkaline phosphatase, which holds a glycosylphosphatidylinositol (GPI) hydrophobic anchor, and the glycosylphosphatidylinositol-specific phospholipase C-solubilized form (PLSAP), lacking the GPI anchor. Similar surface transitions observed for both DMPA and DMPA/PLSAP mixed monolayers indicate that the presence of PLSAP does not promote significant changes in surface packing of the DMPA monolayer. However, PLSAP interacts with the polar portion of the phospholipid even at high lateral compression. The presence of the GPI anchor increases the adsorption of DSAP at a plain air/liquid interface and also enables the penetration of the protein into the DMPA monolayers. The penetration is dependent on both time and surface pressure. Up to 20 mN/m, the surface pressure increases smoothly indicating a diffusion followed by an adsorption process. Above 20 mN/m, after a fast increase, the surface pressure slowly decays to equilibrium values quite close to the initial surface pressures. The results indicate that the molecular packing of the lipid layer drives the enzyme adsorption to the interface either through the GPI anchor or by the polypeptide moiety.     

Uniform molecular analysis using femtosecond laser mass spectrometry

The potential of femtosecond laser time-of-flight mass spectrometry (FLMS) for uniform quantitative analysis of molecules has been investigated. Various samples of molecular gases and vapours have been studied, using ultra-fast ( approximately 50 fs) laser pulses with very high intensity (up to 1.6 x 10(16) Wcm(-2)) for non-resonant multiphoton ionisation/tunnel ionisation. Some of these molecules have high ionisation potentials, requiring up to ten photons for non-resonant ionisation. The relative sensitivity factors (RSF) have been determined as a function of the laser intensity and it has been demonstrated that for molecules with very different masses and ionisation potentials, uniform ionisation has been achieved at the highest laser intensities. Quantitative laser mass spectrometry of molecules is therefore a distinct possibility. Copyright 1999 John Wiley & Sons, Ltd.     

Interpreting early land management through compound specific stable isotope analyses of archaeological soils

Compound specific stable isotope analyses of managed soils using isotope ratio mass spectrometry have been undertaken as a means of determining early land use practices. delta (15)N amino acid signals demonstrate differences between manured grassland, unmanured grassland and continuous cereal cultivation under long-term experimental land use control conditions, with delta (15)N in hydrophobic amino acids providing the most distinctive signals. Analysis of early modern/medieval and of Bronze age anthropogenic soils from Orkney demonstrates that such signals are retained in archaeological contexts. delta (13)C analyses of n- alkanoic acid components of the fossil, Bronze Age, anthropogenic soils suggest a major terrestrial input to these soils, with uniform composition of formation materials. Surficial soils demonstrate the assimilation of isotopically lighter carbon, providing a means of assessing the mobility of the n- alkanoic acids within soils and sediments. Copyright 1999 John Wiley & Sons, Ltd.