Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Enthalpies of formation of transition-metal lanthanide laves phases with the MgCu2 structure

Experimental information and theoretical estimates for the enthalpy of formation at 298 K for the RM₂ (whereM = Fe, Co,orNi andR = La, Ce, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb, or Lu) lanthanide Laves phases with the MgCu₂ (C15) structure are reviewed and discussed in the light of information on lattice parameters and atomic volume. The purpose of the present study is to analyze all the available experimental information on enthalpies of formation for transition-metal lanthanide Laves phases and then plot the data in order to reveal tendencies and allow reliable estimates of data that have not yet been measured. It is concluded that as the lanthanide atomic number increases the enthalpy of formation is almost constant or becomes slightly more negative. The accompanying volume contraction is slightly larger than expected from the lanthanide contraction, 7.6 %. From the so-called Gschneidner correlation one would have expected the contrary behavior, that is, that the bonding strength would become slightly lower when going from LaM₂ to LuM₂. An increase in the transition-metal atomic number has a stronger effect on the enthalpy of formation and atomic volume than an increase in the lanthanide atomic number.     

Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS

Normal-phase HPLC resolution of sn-1,2(2,3)- and x-1,3-DAG generated by partial Grignard degradation from natural TAG was carried out with both (R)-(−) and (S)-(+)-1-(1-naphthyl)ethylurethane derivatives. The diastereomeric sn-1,2- and sn-2,3-DAG derivatives were resolved using two Supelcosil LC-Si (5 μm, 25 cm × 4.6 mm i.d.) columns in series and an isocratic elution with 0.37% isopropanol in hexane at a flow rate of 0.7 mL/min. The DAG were detected by UV absorption at 280 nm and were identified by electrospray ionization MS in the positive ion mode following postcolumn addition of chloroform/methanol/30% ammonium hydroxide (75∶24.5∶0.5, by vol) at 0.6 mL/min. Application of the method to a stereospecific analysis of the molecular species of TAG of rat VLDL showed that the TAG composition of VLDL circulating under basal conditions differs markedly from that of VLDL secreted by the liver during inhibition of serum lipases. The inhibition of serum lipases resulted in a significant proportional decrease in 16∶0 and PUFA and an increase in 18∶0 and oligoenoic FA in the sn-1-position, whereas the FA compositions in the sn-2- and sn-3-positions were much less affected.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Iron Behavior in the Ozonation and Filtration of Groundwater

In Finnish groundwater, the main substances that require treatment are iron and manganese. In addition to this, groundwaters are soft and acidic. Iron removal is usually relatively effective by oxidizing dissolved iron into an insoluble form, either by aeration or chemical oxidization and removing the formed precipitate by sand filtration. Sometimes, if the untreated water contains high amounts of organic matter, problems may arise for iron removal. In Finland, it is quite common that groundwater contains high levels of both iron and natural organic matter, mainly as humic substances. The groundwater of the Kukkala intake plant in Liminka has been found to be problematic, due to its high level of natural organic matter. This research studied the removal of iron from this water by means of oxidation with ozone and filtration. While the oxidation of iron by ozone was rapid, the precipitate particles formed were small, and thus could not be removed by sand and anthracite filtration, and the iron residue in the treated water was more than 2 mgL^?1. And while the filtration was able to remove iron well without the feed of ozone, the iron residue in the treated water was only 0.30 mgL^?1. In this case, iron was led to the filter in a bivalent dissolved form. So, the result of iron removal was the best when the sand/anthracite filter functioned largely as an adsorption filter.