Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.     

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT–SPD–FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A₁₆₅. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT–SPD–FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT–SPD–FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)–SPD–FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.     

Intermediates in the Methanol-to-hydrocarbons (MTH) Reaction: A Gas Phase Study of the Unimolecular Reactivity of Multiply Methylated Benzenium Cations

In order to reach a deeper insight into the reaction mechanism of the zeolite catalyzed methanol to hydrocarbons reaction (MTH), the proposed reaction intermediates, i.e., a series of multiply methyl-substituted benzenium ions has been generated in the gas phase by chemical ionization. The fragmentations of the corresponding long-lived (metastable) ions have been investigated. While expulsion of H₂ dominates for the lower homologues, elimination of methane dominates for the higher homologues, accompanied by increasing amounts CH₃^·. Loss of larger fragments relevant to the MTH-reaction, in particular ethene, propene and even butene, is also observed in minor amounts. This latter finding is consistent with a proposed reaction cycle in the MTH reaction known as the paring mechanism, and the feasibility of this mechanism has thus been demonstrated. The metastable gas-phase ions studied here are considerably more energetic than those residing in a zeolite catalyst, but they were found to decompose with markedly higher selectivity towards alkenes as compared to those activated by collision-induced dissociation (CID).     

Binding-time analysis for Standard ML

We present an efficient base algorithm for binding-time analysis based on constraint solving and the union-find algorithm. In practice it has been used to handle all of Standard ML except modules and we show the principles of how constraints can be used for binding-time analysis of Standard ML; in particular we show how to binding-time analyse nested pattern matching. To the best of our knowledge no previous binding-time analysis has treated nested pattern matching.The present work was conducted while the author was at DIKU.     

Effect of Hydrotropic Substances on the Complexation of Clotrimazole with β-Cyclodextrin

The phase diagrams of clotrimazole/β-cyclodextrin (β-CD) in phosphate buffer, pH = 7.1, containing 0.5 M of various hydrotropic agents were constructed. The water structure disruptors, urea and nicotinamide, increased the intrinsic solubility of the antimycotic drug clotrimazole while the water structure forming agents, sorbitol and fructose, decreased the solubility. Concerning the complex constant between clotrimazole and β-CD, it was the other way around. The connection between the slopes of the phase diagrams, the intrinsic solubility of clotrimazole and the complex constant was discussed. Nicothamide decreased the solubility of β-CD in the buffer solution. The results reported in this study are in disagreement with the claim that addition of water structure forming agents to cyclodextrin solutions can be used to increase the total solubility of drugs     

Structural and electrical properties of perovskite ruthenate-based lead-free thick film resistors on alumina and LTCC

CaRuO₃ perovskite-based lead-free thick-film resistors (TFRs) were prepared on 96%-alumina and Low Temperature Co-fired Ceramic (LTCC) substrates. The microstructure evolution, possible interactions, and electrical properties of resistors were investigated. The hot and cold TCR values of all the resistors were measured in the temperature range (+20 to +120 ^∘C) and (+20 to −120 ^∘C), respectively. TFRs with 25% by vol. of CaRuO₃ on alumina exhibit a sheet resistance R_s = 5kΩ/sq. with hot and cold TCR of 225 and 470 ppm/^∘C respectively, whereas the same composition gives 1.2 kΩ/sq., 16.5 kΩ/sq. and 0.7 kΩ/sq. for co-fired, post-fired resistors on LTCC and buried resistors, respectively. The hot (HTCR) and cold (CTCR) values were evaluated; HTCR = 190 ppm/^∘C and CTCR = 314 ppm/^∘C were found for co-fired structures; HTCR = 216 ppm/^∘C and CTCR = 205 ppm/^∘C for post-fired samples and HTCR = 520 ppm/^∘C and CTCR = 350 ppm/^∘C for buried in LTCC structures.     

Visual scoring of clots in foremilk

The necessary unequivocal and generally accepted definitions of normal and abnormal milk are not available. A precise definition is needed in order for companies to develop sensors to detect and sort abnormal milk at the time of milking. Experts at a workshop defined abnormal milk to be that from cows whose foremilk had changed in homogeneity or was coloured by blood. The objectives of this paper were: firstly, to explore how different groups of people scored the appearance of foremilk; and secondly, to develop a method suitable as an objective reference for testing of manual and automatic detection systems. Consumers, farmers and advisors did not agree on the visual appearance of normal, watery, clotty milk, or milk with blood, and experience is needed to score the visual appearance of foremilk correctly. It seems reasonable to expect a sensitivity of at least 70% for detection of abnormal milk during foremilking. Filter sizes 0.05, 0.07, 0.1, 0.2, 0.5, 1.0, and 2.0 mm were used to filter milk from cows with visually abnormal foremilk. If clots appeared in the foremilk, clots appeared on all size filters, but the filter with pore size 0.1 mm was the easiest to read and work with. The filter method is not reliable in identifying quarters with watery, yellowish, or bloody milk, whereas the method seems consistent, and at least as good as scoring of visual appearance in finding clots in the milk. Clots should show clearly on the filter to be counted as abnormal milk. All clinical cases with clots in the foremilk can be found on the filter and such cases have high somatic cell count (SCC). Both trained and untrained persons using the filter method can score normal and abnormal foremilk with a high specificity (>90%) and a high sensitivity (>80%). The filter method is recommended as a reference for scoring the homogeneity of foremilk.