Unfolding kinetics of beta-lactoglobulin induced by surfactant and denaturant: a stopped-flow/fluorescence study

The beta-->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I(1)) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I(2)) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.     

Analysis of indentation: implications for measuring mechanical properties with atomic force microscopy.

Indentation using the atomic force microscope (AFM) has potential to measure detailed micromechanical properties of soft biological samples. However, interpretation of the results is complicated by the tapered shape of the AFM probe tip, and its small size relative to the depth of indentation. Finite element models (FEMs) were used to examine effects of indentation depth, tip geometry, and material nonlinearity and heterogeneity on the finite indentation response. Widely applied infinitesimal strain models agreed with FEM results for linear elastic materials, but yielded substantial errors in the estimated properties for nonlinear elastic materials. By accounting for the indenter geometry to compute an apparent elastic modulus as a function of indentation depth, nonlinearity and heterogeneity of material properties may be identified. Furthermore, combined finite indentation and biaxial stretch may reveal the specific functional form of the constitutive law--a requirement for quantitative estimates of material constants to be extracted from AFM indentation data.     

Environmental assessment in an industrial area of Portugal

The region of Lisbon and south of Lisbon (Sado estuary) is densely industrialized, and, therefore, air pollution should be studied in a more detailed scale there. The topography of the Sado estuary region and the predominant wind direction from the northwest contribute to the influence in this region of the industries located in the north. The region selected includes a fuel-fired power station. Transplants of the lichenParmelia sulcata Taylor were suspended in nylon bags within a rectangle 15 km wide and 25 km long on a grid 2.5 km × 2.5 km, centered in the power station. In each of the 47 sites, 2 sets of 4 transplants each were hung. Care was taken (1) in covering the two sets with a polyethylene roof to prevent leaching of elements in the lichen, (2) in building a hanging system that could rotate according to the wind direction, and (3) in orienting one set toward the wind and the other set opposite the wind. For a 1-yr period and every 3 mo, one transplant of each set is collected. In this work, the results of the first campaign (after 3 mo suspension) obtained by instrumental neutron activation analysis and proton-induced X-ray emission are shown. Some elemental contents are mapped and discussed.     

Indigenous parasitoids (Hym., Chalcidoidea) of Phyllocnistis citrella Stainton (Lep., Gracillariidae) in Jaguariúna, São Paulo State, Brazil: preliminary results

Abstract: The citrus leafminer, Phyllocnistis citrella Stainton, is native to Asia and was detected for the first time in Brazil in 1996. The objectives of this study were to conduct a survey of parasitoids attacking citrus leafminer in Jaguariúna, one of the citrus production regions of São Paulo State, Brazil, and to determine whether indigenous species were providing effective control of this pest species. The collections of new leaves were made weekly at citrus groves in Jaguariúna from October 1996 to October 1997. Of the six indigenous parasitoids which were found, Galeopsomyia fausta LaSalle was the most abundant parasitoid of P. citrella in this region, accounting for more than 90% of the specimens collected. Thus, this species is a serious candidate for biological control of the citrus leafminer in this region of Brazil. Other species collected were Horismenus sp., Cirrospilus 'sp. C', Elasmus sp., Eupelmus sp. and Conura (Ceratosmicra) sp. The mean percentage of parasitism was 39.28% (6.19−86.21%) in unsprayed orchards and 21.38% (1.33−56.63%) in orchards that were occasionally treated with fungicides, acaricides and insecticides Temik and Dipterex.     

Immobilization of glucose oxidase on thin-film gold electrodes produced by magnetron sputtering and their application in an electrochemical biosensor

An electrochemical biosensor is described consisting of a thin-layer gold film electrode prepared by cathodic sputtering using a poly(vinyl chloride) sheet as substrate, with voltammetric behaviour comparable to that of conventional polycrystalline gold electrodes, coated with the hydrolysed copolymer hydroxyethyl methacrylate-co-methyl methacrylate onto which glucose oxidase was immobilized. The mechanical properties of the plastic foil substrate permit easy construction of circular-shaped electrodes which were employed as working electrodes for batch injection analysis. The electrochemical biosensor fabrication is inexpensive and can be used as disposable enzyme sensor for the detection of hydrogen peroxide. The biosensor was tested for the determination of glucose in serum and a good correlation was obtained with the measurement using the electrochemical and the spectrophotometric methods.     

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed. Received: February 10, 1998 / Accepted: January 11, 1999