Iron gall ink-induced corrosion of cellulose: aging,degradation and stabilization. Part 2: application on historic sample material

Degradation of cellulose in historic paper by iron gall ink is a synergistic process of both, acid hydrolysis caused by acidic ink ingredients and oxidation catalyzed by free iron and/or copper ions. The interplay of both reactions was studied according to the CCOA method on historic paper samples. Only minute amounts (few mg) of the samples were required to obtain profiles of naturally present and oxidatively introduced carbonyl groups, which was done by group-selective fluorescence labeling in combination with determination of the molecular weight distribution by GPC-MALLS. In the present study naturally occurring degradation pathways in historic sample papers have been investigated. Different extents of oxidatitive degradation were shown for paper with and without ink. A typical pattern of the molecular weight distribution in naturally aged papers was identified, the peculiar feature being a distinctive shoulder in the region of low molecular weight, roughly between 25,000 and 5,000 g/mol corresponding to a DP between 150 and 30. This pattern was a typical attribute of degraded natural samples: any artificial aging procedures aimed at modeling natural aging processes must thus attempt to reproduce this feature. Although the historic samples had been more severely oxidized than model papers, the inhibition of further oxidation and hydrolysis by the calcium phytate/hydrogen carbonate treatment was evident and could be proven for the first time on the molecular level. Also on plain paper without ink application the oxidation was suppressed and the molecular weight was stabilized on a high level.     

Iron gall ink-induced corrosion of cellulose: aging,degradation and stabilization. Part 1: model paper studies

Cellulose in historic paper documents is often damaged by the writing media used, especially iron gall ink or copper pigments. Degradation induced by iron gall ink is suggested to be a synergistic process comprising both hydrolytic and oxidative reactions. These processes were studied on very low sample amounts according to the CCOA and FDAM method, i.e. by fluorescence labeling of carbonyl and carboxyl groups in combination with GPC-MALLS, respectively. This study focused on preventive means to stop the deterioration induced by iron gall ink of cellulose and to prevent further damage, keeping in mind that a suitable conservation treatment has to hinder both, hydrolytic and oxidative processes, at the same time. A combination of the complexing agent calcium phytate and calcium hydrogencarbonate in aqueous solution was proved to give optimum results. To gain insight into long term stability, an aging step was performed after treatment and different ink modifications were tested. Recording the molecular weight distributions and the carbonyl group content over time GPC analysis verified for the first time the preventive effect of this treatment. This effect was not only seen for the ink-covered areas, but extended also to areas remote from the ink lines. Ink containing copper ions responded equally positively to the calcium phytate/hydrogencarbonate treatment as the iron gall ink papers did. Gelatine, sometimes used in a similar way due to an alleged cellulose-stabilizing effect did not have a beneficial influence on cellulose integrity when metal ions were present.     

Kinetic properties of hexameric tyrosinase from the crustacean Palinurus elephas

Tyrosinases catalyze hydroxylation of monophenols to o-diphenols and their subsequent oxidation to o-quinones, whereas catecholoxidases catalyze only the latter reaction. Both enzymes occur in all organisms and are Type 3 copper proteins that perform the first steps of melanin formation. In arthropods, they play an essential role in the sclerotization of the exoskeleton. Very few phenoloxidases are characterized structurally or kinetically and the existence of an actual tyrosinase activity has not been demonstrated in most cases. Here we present for the first time a complete kinetic characterization of a tyrosinase from a crustacean (Palinurus elephas) including the influence of inhibitors. In contrast to most tyrosinases which are monomeric or dimeric, this tyrosinase occurs as a hexamer. However, the data did not indicate cooperativity in steady-state kinetics for the two substrates used, the monophenol tyramine and the diphenol dopamine. Mimosine as well as phenylthiourea (PTU) inhibited both monophenolhydroxylase and diphenoloxidase activity. Inhibition by mimosine was competitive, whereas PTU was a noncompetitive inhibitor. Furthermore, for the diphenolase activity substrate inhibition was observed, which was apparently abolished by adding PTU. These observations lead to the hypothesis that a secondary, allosteric binding site exists, which binds dopamine and PTU and reduces the catalytic activity.     

Bioinformatical evaluation of modified nucleosides as biomedical markers in diagnosis of breast cancer

It is known that patients suffering from cancer diseases excrete increased amounts of modified nucleosides with their urine. Especially methylated nucleosides have been proposed to be potential tumor markers for early diagnosis of cancer. For determination of nucleosides in randomly collected urine samples, the nucleosides were extracted using affinity chromatography and then analyzed via reversed phase high-performance liquid chromatography (HPLC) with UV-detection. Eleven nucleosides were quantified in urine samples from 51 breast cancer patients and 65 healthy women.The measured concentrations were used to train a Support Vector Machine (SVM) and a k-nearest-neighbor classifier (k-NN) to discriminate between healthy control subjects and patients suffering from breast cancer. Evaluations of the learned models by computing the leave-one-out error and the prediction error on an independent test set of 29 subjects (15 healthy, 14 breast cancer patients) showed that by using the eleven nucleosides, the occurrence of breast cancer could be forecasted with 86% specificity and 94% sensitivity when using an SVM and 86% for both specificity and sensitivity with the k-NN model.     

Protein G-liposomal nanovesicles as universal reagents for immunoassays

To improve the antigen-binding activity of liposome-coupled antibodies and to develop universal liposomal nanovesicles for immunoassays, protein G was conjugated to dye-loaded liposomal nanovesicles for the preparation of immunoliposomes. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), a heterobifunctional cross-linker, was used to modify protein G for conjugation to the liposomal nanovesicles. Liposome immunosorbent assays were used to evaluate the binding ability of protein G after sulfo-SMCC modification, to optimize the protein G density on the liposome surface and to determine the amount of IgG binding to the protein G-liposomal nanovesicles. Test strips coated with a narrow zone of antibodies were used to show the successful conjugation. Immunomagnetic beads were used to demonstrate the feasibility of protein G-tagged universal liposomal nanovesicles for immunoassays. Results indicate that the Fc-binding capacity of protein G decreased by only 5.3% after sulfo-SMCC modification. Antibodies were easily conjugated to universal protein G-liposomal nanovesicles in 30 min. The conjugates (protein G-immunoliposomes) were successfully used in immunomagnetic bead assays for the detection of Escherichia coli O157:H7 with a detection limit of approximately 100 CFU/ml. This work demonstrated that protein G-liposomal nanovesicles are a successful universal reagent for easily coupling antibodies in an active orientation on the liposome surface for use in immunoassays.     

First generation and trapping of a dehydrometallophthalocyanine starting from triazole-functionalized zinc phthalocyanine

Direct 1N-amination of the triazole-fused zinc phthalocyanine 2 and oxidation of the formed amino derivative 3 resulted in the generation of the very reactive intermediate, the dehydrometallophthalocyanine 4, which was not known previously. The latter was trapped in situ with different dienes, for example, furan, tetraphenylcyclopentadienone, and anthracene to form the corresponding Diels-Alder adducts. The products were characterized by 1H and 13C-dept135 NMR, and UV/Vis spectroscopy, MALDI-TOF mass spectrometry, and elemental analysis, which are fully in agreement with their structure. The developed synthetic procedure opens a simple and versatile pathway towards unsymmetrical peripheral modification of phthalocyanines, which is readily applicable to the micromol scale and is important for the design of new interesting Pc-based systems.     

Variations in Physicochemical Properties and Bioconversion Efficiency of <Emphasis Type="Italic">Ulva lactuca</Emphasis> Polysaccharides After Different Biomass Pretreatment Techniques

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.     

Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo

We report here the generation of mutants of the human O(6)-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter molecules. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O(6)-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O(6)-benzylguanine derivatives, the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small molecules and therefore should become important tools for studying protein function in living cells. In addition to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O(6)-benzylguanine.