Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2μ-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 × HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.     

Analysis of similarity of electrophoretic patterns in mRNA differential display

We present a novel method of statistical analysis for the comparison of electrophoretic data. The method is based on the squared Euclidian distance of normalized signal data vectors of electrophoretic lanes. The differences in the electrophoretic patterns are evaluated by a statistical test based on Hubert's statistics which measures the significance of the signal grouping. We demonstrate the validity and applicability of the method in a large data set derived from automated fluorescent mRNA differential display analysis of the expression of acute-phase proteins during experimental Escherichia coli infection in mice. The current testing method is capable of finding theoretically similar natural groupings to be similar in a statistically significant way whereas theoretically dissimilar or random groupings can be recognized to be artifactual. We also show how the calculated pairwise signal distances can be utilized in methodological problem solving. These analytical methods can be applied to the study of other related problems of similarity analysis of electrophoretic patterns, and also provide useful tools for the development of automated recognition of differentially expressed mRNAs.     

Antibacterial activity of methylated chitosan and chitooligomer derivatives: Synthesis and structure activity relationships

The purpose of this study was to synthesize series of methylated chitosaccharide derivatives, possessing various degree of methylation, and to determine their structure activity relationship (SAR) with regard to their antibacterial effect against Staphylococcus aureus. Chitosan polymer and chitooligomers were used as starting materials and were methylated by reaction with methyl iodide. Depending on the reaction conditions the degree of N-quaternization ranged from 0% to 74%, with varying degree of N,N-dimethylation, N-monomethylation and O-methylation. More selective N-quaternization could be obtained with protection group strategy. At pH 5.5 the chitosaccharide polymers and their methylated derivatives were active against S. aureus with minimal inhibitory concentration (MIC) ranging from 16 to 512 μg/mL. At pH 7.2 the non-quaternized derivatives were inactive but their highly N-quaternized derivatives showed MIC as low as 8 μg/mL. The chitooligomers, as well as their derivatives, were inactive at both pH’s. The SAR studies revealed that N-quaternization was mainly responsible for the antibacterial effects at pH 7.2, whereas it did not contribute to the antibacterial activity under acidic conditions.     

The identification of vicinally substituted cyclohexane isomers in their mixtures by 1H and 13C NMR spectroscopy

The radical addition reactions of organobromine compounds, XBr (X = CH2COOMe, PhCH2, CHBr2 and CCl3) with cyclohexene afforded mixtures of cis/trans isomer pairs of 1-X-2-Br-cyclohexanes. In addition to benzyl benzoyloxy derivatives are formed also, when benzoyl peroxide is used as an initiator. Owing to the great difficulties in separating these cis/trans isomer pairs, they are identified directly in their mixtures by NMR spectroscopy. In addition to one-dimensional (ID) 1H, proton decoupled 13C and DEPT-135, also two-dimensional (2D) 13C-13C INADEQUATE as well as 1H-13C HMQC experiments have been used in assigning the signals of each compound in their mixtures. The identification of each isomer was based on comparison of experimental 3J(H,H) coupling constants with theoretical ones based on the well-known Karplus type relationship. The more stable conformation for each isomer was estimated using the semiempirical AM1 molecular orbital method. The calculations support the isomer pair elucidations.     

URB754 has no effect on the hydrolysis or signaling capacity of 2-AG in the rat brain

Previous studies indicate that in brain tissue the endocannabinoid 2-AG is inactivated by monoglyceride lipase (MGL)-catalyzed hydrolysis, and a recent report has indicated that MGL activity could be specifically inhibited by URB754 . In the present study, URB754 failed to inhibit 2-AG hydrolysis in rat brain preparations. In addition, brain cryosections were employed to assess whether URB754 could facilitate the detection of 2-AG-stimulated G protein activity. Nevertheless, whereas pretreatment with PMSF readily allowed detection of 2-AG-stimulated G protein activity, URB754 was ineffective. In contrast to previous claims, brain FAAH activity was also resistant to URB754. Thus, in our hands URB754 was not able to block the endocannabinoid-hydrolyzing enzymes and cannot serve as a lead structure for future development of MGL-specific inhibitors.     

Electron impact induced fragmentations of some 2,2-disubstituted 1,3-oxathiolanes

The electron impact induced fragmentations of nine 2,2-disubstituted 1,3-oxathiolanes have been studied by means of exact mass measurement and metastable ion analysis. The ring cleavage almost always takes place so that the C(2)? S and C(5)? O bonds are broken, leading to the most stable products. The nature of the substituents determines the primary fragmentations of molecular ions. Ring cleavage is important only if both substituents are alkyl groups or if the carbon attaching to the ring has an alkyl character. The loss of the substituent becomes the most favourable process if it is attached to the ring through the electron-deficient carbon atom.     

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.

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Figure A single biofunctionalized nanoparticle was attached to the outer tip apex and the binding behavior of the nanoparticle in a sandwich-type immunoassay, A) without analyte, B) with analyte and C) saturating analyte concentration, was recorded using dynamic force spectroscopy in millisecond timescale. The setting allowed measurement of the association speed of nonspecific binding.

     

Diffusion of Tracer in Altered Tonalite: Experiments and Simulations with Heterogeneous Distribution of Porosity

Numerical time-domain-diffusion simulations were used for studying the diffusion behavior of tracer molecules in rock matrix with homogeneous and heterogeneous porosity. As the heterogeneous sample in these simulations, a 3D tomographic image of altered tonalite was used, in which the mineral components and the pores resolved by X-ray microtomography were represented by their respective intragranular porosities determined previously by the ¹⁴C-PMMA method. The apparent diffusion coefficient of a tracer in altered tonalite was determined experimentally, and was then used in the simulations. In the altered tonalite analyzed, inclusion of heterogeneity in the porosity increased the diffusion coefficient by 16 %. Altered and pristine feldspar was the main mineral component in the sample (72 %), and it also provided the dominant contribution to tracer diffusion, explaining alone 52 % of the diffusion coefficient. The large pores resolved by microtomography (6 %) and altered and pristine mica (22 %) gave an equal contribution to the diffusion coefficient. The simulation method applied was also validated by comparing the results to both an analytical and a numerical solution to the diffusion equation in a homogenous medium. In addition, the method was compared to discrete-time random-walk simulations in the case of randomly placed overlapping spheres.