Rute I.S. Romão

l‐Arabinose transport and catabolism in yeast

Two yeasts, Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012, which show rapid growth on l‐arabinose and very high rates of l‐arabinose uptake on screening, were selected for characterization of l‐arabinose transport and the first steps of intracellular l‐arabinose metabolism. The kinetics of l‐arabinose uptake revealed at least two transport systems with distinct substrate affinities, specificities, functional mechanisms and regulatory properties. The l‐arabinose catabolic pathway proposed for filamentous fungi also seems to operate in the yeasts studied. The kinetic parameters of the initial l‐arabinose‐metabolizing enzymes were determined. Reductases were found to be mostly NADPH‐dependent, whereas NAD was the preferred cofactor of dehydrogenases. The differences found between the two yeasts agree with the higher efficiency of l‐arabinose metabolism in C. arabinofermentans. This is the first full account of the initial steps of l‐arabinose catabolism in yeast including the biochemical characterization of a specific l‐arabinose transporter.

Schizophrenic Behavior of a Thermoresponsive Double Hydrophilic Diblock Copolymer at the Air−Water Interface

The thermoresponsive behavior of the rhodamine B end-labeled double hydrophilic block copolymer (DHBC) poly(N,N-dimethylacrylamide)-b-poly(N,N-diethylacrylamide) (RhB-PDMA(207)-b-PDEA(177)) and the 1:1 segmental mixture of PDEA and rhodamine B end-labeled PDMA homopolymers was studied over the range of 10-40 degrees C at the air-water interface. The increase in collapse surface pressure (second plateau regime) of the DHBC with temperature confirms the thermoresponsiveness of PDEA at the interface. The sum of the pi-A isotherms of the two single homopolymers weighted by composition closely follows the pi-A isotherm of the DHBC, suggesting that the behavior of each block of the DHBC is not influenced by the presence of the other block. Langmuir-Blodgett monolayers of DHBC deposited on glass substrates were analyzed by laser scanning confocal fluorescence microscopy (LSCFM), showing schizophrenic behavior: at low temperature, the RhB-PDMA block dominates the inside of bright (core) microdomains, switching to the outside (shell) at temperatures above the lower critical solution temperature (LCST) of PDEA. This core-shell inversion triggered by the temperature increase was not detected in the homopolymer mixture. The present results suggest that both the covalent bond between the two blocks of the DHBC and the tendency of rhodamine B to aggregate play a role in the formation of the bright cores at low temperature whereas PDEA thermoaggregation is responsible for the formation of the dark cores above the LCST of PDEA.

Incorporation of β-lactoglobulin in monolayers of dioctadecyldimethylammonium bromide studied by Brewster angle microscopy

The deposition of pure bovine β-lactoglobulin (βLG) monolayer or the incorporation in a dioctadecyldimethylammonium bromide (DODAB) monolayer, were studied by π–A measurements at the air–water interface and by direct visualization of the interface by BAM. The co-spreading of the monolayer material dissolved in a mixed volatile solvent (chloroform+ethanol) was selected based on reproducible data and minimum consumption of protein. Conformational changes induced by the mixed solvent on the native structure of βLG, investigated by circular dicroism, disappear after the solvent evaporation at the interface. The π–A isotherms suggest and BAM images confirm, that, at low surface pressures, βLG is incorporated in the liquid expanded monolayer of DODAB, while at the plateau near 30 mN m−1 the protein is squeezed out into micro-domains partially immersed in the subphase and strongly adsorbed under the DODAB layer. The topography of DODAB/yβLG mixed films changes with surface pressure and number of protein residues, y, incorporated per DODAB molecule. BAM observation shows that at low y DODAB molecules dominate the topography of monolayer at the interface.

Microphase separation in mixed monolayers of DPPG with a double hydrophilic block copolymer at the air-water interface: a BAM, LSCFM, and AFM study.

Phase separation and interactions in mixed monolayers of dipalmitoylphosphatidylglycerol (DPPG) with the rhodamine B end-labeled double-hydrophilic block copolymer (DHBC), poly(N,N-dimethylacrylamide)-block-poly(N,N-diethylacrylamide) (RhB-PDMA(207)-b-PDEA(177)), was studied at the air-water interface. Surface pressure versus area isotherms indicate that both components behave almost independently. Brewster angle microscopy (BAM) images show a random distribution of liquid condensed (LC) domains of DPPG in an apparent homogeneous matrix of DHBC, excluding the macroscopic phase separation. The laser scanning confocal fluorescence microscopy (LSCFM) of the rhodamine dye at the end of the PDMA chain showed how the DHBC is distributed in Langmuir-Blodgett (LB) mixed monolayers. The high spatial resolution of atomic force microscopy (AFM) combined with the LCSFM images indicates that DHBC incorporates in the expanded phase of DPPG to form mixed domains, being excluded from the condensed regions. Upon compression, nanosized LC domains of DPPG nucleate inside the mixed domains corralled in the nanopatterning of pure DHBC. The negatively charged polar group of DPPG inhibits rhodamine aggregation, while the long polymer chains promote the formation of corralled nanodomains of DPPG in two dimensions.

Behaviour of the water-soluble meso-tetra(4-methylpyridyl)porphine in mixed monolayers and in Langmuir–Blodgett films

The water-soluble tetracationic meso-tetra(4-methylpyridyl)porphine (PO2) was incorporated in mixed Langmuir monolayers (LM) with the anionic surfactant sodium hexadecylsulfate (SHS) and stearic acid (SA). The stability of the monolayer and the molecular packing of PO2 vary with the surface pressure and amount of stearic acid in the monolayer. The aggregation or tilting of PO2, occurring at the long transition during the compression run at the air–water interface, is reversible for all the ternary systems studied (PO2/SHS/SA). The organization of PO2 in LM, deduced from the π–A isotherms, is confronted with the spectroscopic data of LB monolayers and multilayers on silica substrates. At surface pressures below the long transition only one layer transfers onto the solid substrate whatever the number of vertical transfer strokes performed, while at high surface pressures LB films with several layers were formed. All absorption spectra of LB films exhibit a Soret maximum at λ1 ≈ 433 nm, which is ascribed to a nearly flat conformation of the PO2 ring. LB monolayers and multilayers of systems PO2/4SHS/4SA and PO2/4SHS/8SA, formed at high π(50 mN m−1), show an additional absorption band centred at λ2 ≈ 405 nm, which may account for a nonplanar conformation of PO2 with the pyridinium rings out of the porphine plane. The results suggest that the planar form of PO2 is imposed in the first layer by direct contact in a parallel orientation to the solid substrate, while the nonplanar form may coexist or even prevail in upper layers of LB films formed at high π.