Peer assessment as a method for measuring harmful internet use.

Harmful Internet use (HIU) describes unintended use of the Internet. It could be both self-harm and harming others. Our research goal is to develop a more accurate method for measuring HIU by this novel peer assessment. As such, it may become, with our call for more research, a paradigm shift supplementing every rating scale or other type of Internet use assessment. In addition to classic statistical analysis, structural equations have been employed. Results indicate that the true positive rate (TPR) is substantially higher than assessed in other studies.•Peer assessment improvement.•AUC for ROC was computed to establish cut-off points for the used scale.•Results obtained by the Structural Equation model indicate that parental care has a moderate influence on subjects' attempts to fight HIU.

The impact of lipid oxidation on the functioning of a lung surfactant model.

Apart from being responsible for sufficient pulmonary compliance and preventing alveolar collapse, lung surfactant (LS) also forms the first barrier for uptake of inhaled pathogens. As such it is susceptible to damage caused by various deleterious compounds present in air, e.g. oxidants capable of oxidizing unsaturated LS lipids. This study examines the consequences of oxidizing 20% of unsaturated lipids in an LS model: a mixed 1 : 1 DPPC : POPC monolayer. POxnoPC (1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine) is considered as the main oxidation product. Experimental surface pressure-area isotherms and polarization-modulation infrared reflection-absorption spectroscopy are employed to probe changes in the macroscopic properties of the unsaturated lipid monolayer induced by oxidation. Microscopic details of the influence of oxidation on the monolayer's phase behavior are elucidated by molecular dynamics simulations at varying lipid packing. We demonstrate that unsaturated lipid oxidation shifts the isotherm towards larger areas and advances monolayer collapse. This is caused by a reversal of the oxidized sn-2 chains of POxnoPC towards the subphase, driven by electrostatic interactions between the aldehyde, glycerin, and water. Increased lipid bulkiness, hindered transition to the LC phase, and transfer of oxidized chain terminals to the subphase have been identified as the most troublesome consequences of this process. They result in the reduction of monolayer stability and its capability to withstand high surface pressures. This may lead to uncontrolled and irreversible loss of lipids from the surface.

Derivatives of glutamic acid as new surfactants.

Starting from glutamic acid, different types of surfactants have been synthesised by using original trimodular strategies. Monosubstituted zwitterionic amides of glutamic acid obtained with excellent yields show good surface activity. The grafting of a second hydrophobic side-chain leads to bicatenar cationic surfactants or to disubstituted nonionic cyclic compounds. In order to reduce the hydrophobic character of the bicatenar surfactants, a second synthetic method has been developed, allowing the introduction of a polar sugar group into these molecules. The surfactant properties of several of the products have been determined by physico-chemical methods such as surface tension measurements and compression isotherm studies by means of a Langmuir balance.

Formation and properties of Langmuir and Gibbs monolayers: a comparative study using hydrogenated and partially fluorinated amphiphilic derivatives of mannitol.

The synthesis and surface behavior of a series of nine new hydrogenated nonionic surfactants and their fluorinated analogs, derived from D-mannitol are described. Adsorption monolayers (Gibbs monolayers) were studied by surface pressure (H) measurements as a function of time. For the spread monolayers (Langmuir monolayers), the measurements of surface pressure versus molecular area (A) were performed. For the most hydrophobic amphiphiles at low concentrations, the adsorption at the air/water interface from the bulk solution required extremely long times to attain equilibrium. The A values for two compounds which could be studied in both adsorbed and spread monolayers provided data allowing a direct comparison of the properties of the two types of films formed at the air/water interface. In spite of different mechanisms of formation of Langmuir and Gibbs monolayers, their characteristic parameters were identical, proving the equivalence of these two types of structures.

Synthesis and properties of two new liquid crystals: an analytical and thermodynamic study.

Synthesis, analytical performances, thermodynamic and surface properties of two new liquid crystals substituted with poly(ethylene oxide) chains are described. The first of them is N,N'-diphenyl-[4-[2,3,4-tri[2-(2-metoxyethoxy)ethoxy]benzylidene]i mine]piperidine (LC1) and the second is 2-hydroxy-3-methyl-4-[4-[2-(2-butoxyethoxy)ethoxy]] 4'-[4-[2-(2-butoxyethoxy)ethoxy]styryl]azobenzene (LC2). The nematic ranges of the two liquid crystals, determined by differential scanning calorimetry do not interfere. The analytical and thermodynamic studies of LC1 and LC2 in the solid, nematic and liquid state were done using a series of appropriate solutes. Comparison of the analytical performances shows a better efficiency in the nematic state.

Self-assembly of chlorophenols in water.

In saturated solutions of some di- and trichlorophenols, structures with complex morphologies, consisting of thin, transparent sheets often coiling into helices and ultimately twisting into filaments, were observed under the optical microscope. Freeze-fracture electron microscopy, x-ray diffraction, phase diagrams, and molecular modeling were performed to elucidate the observed phenomena. Here, we present evidence that the chlorophenols studied, when interacting with water, self-assemble into bilayers. The fact that some chlorophenols form the same supramolecular structures as those described previously for structurally nonrelated surfactants sheds light on the mechanisms of self-assembly.

In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases.

The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.

Phosphate-binding sites in phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The two anion-binding sites of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GraP-DH), the Ps and Pi sites, were originally proposed by Moras et al. [Moras, D., Olsen, K.W., Sabesan, M.N., Buehner, M., Ford, G.C. & Rossmann, M. G. (1975) J. Biol. Chem. 250, 9137-9162] to bind the C3 phosphate of the glyceraldehyde 3-phosphate and the inorganic phosphate respectively. Ps site mutants T179A, and T179M, and R231L, and the Pi site mutants T150A and T208 of the Bacillus stearothermophilus GraP-DH were constructed by site-directed mutagenesis and their kinetic properties were determined and compared with those of mutants R195L and R231G, already described [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260-3265]. Taking advantage of the opportunity to study both the oxidoreduction and the phosphorylation step independently and the fact that the phosphorylation becomes rate determining for most of the mutants, the relative energetic contribution of each mutated amino acid to the phosphorylation step was evaluated. It was concluded that (a) Ps amino acids contribute more than the Pi amino acids to the stabilisation of the transition state relative to the ground state and (b) the side chain of arginine contributes more than that of the threonine residue. It was also concluded that the differences observed in the efficiency of the phosphorylation step for Ps and Pi mutants is a consequence of the orientation of the thioester bond of the thioacyl] intermediate relative to the attacking inorganic phosphate and not of a change in the intrinsic electrophilic property of the thioacyl intermediate. Furthermore, the kinetic results on the overall steps leading to the acyl-enzyme formation provided supplementary evidence that the C3 phosphate moiety of the glyceraldehyde 3-phosphate interacts with the Pi site during these steps and thus are consistent with the findings of Skarzynski et al. [Skarzynski, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol Biol. 193, 171-183] and Corbier et al. [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260-3265] that recommended the reconsideration of the first definition of the Ps and Pi sites.

Stereoselective hydrolysis of triglycerides by animal and microbial lipases.

In the present paper, a study on the stereoselectivity of 25 lipases of animal and microbial origin towards homogeneous prochiral triglycerides is presented. All the lipases tested catalyse the hydrolysis of the chemically alike but sterically nonequivalent ester groups in trioctanoin and triolein with different degrees of stereobias, depending on the fatty acyl chain length of the substrate (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990). Hydrolysis of the sn-2 ester group is catalysed by very few lipases and only Candida antarctica A shows a clear preference for this position. Most of the lipases investigated (12 with trioctanoin and 16 with triolein) showed a preference for the sn-1 position. Using trioctanoin as substrate we observed a total stereoselectivity for position sn-1 with Pseudomonas sp. and Pseudomonas aeruginosa and for position sn-3 with Candida antarctica B. This was not the case with triolein as substrate. Among the 23 lipases studied here and the other two lipases described previously (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990), 17 show a higher stereoselectivity with trioctanoin than with triolein. With guinea pig pancreatic lipase and with three mold lipases (Geotrichum candidum M, Geotrichum candidum A, and Candida antarctica B), the preference switches from sn-3 to sn-1 when the acyl chain length increases from eight to 18 carbon atoms. The main conclusion to emerge from the present study is that the specific stereopreference of each lipase for a given substrate under given lipolytic conditions can be said to be its fingerprint.

Controlling lipase stereoselectivity via the surface pressure.

In the present study, the stereoselectivity of Rhizomucor miehei lipase, lipoprotein lipase, Candida antarctica B lipase, and human gastric lipase towards racemic dicaprin spread as a monolayer at the air-water interface was investigated. For this purpose we have developed a method with which the enantiomeric excess of the residual substrate can be measured in monomolecular films. The stereoselectivity, which is one of the main aspects of enzymic catalysis, was found to depend on the surface pressure of the substrate. With all four lipases tested, low surface pressures enhanced the stereoselectivity while decreasing the enzymes' catalytic activity.

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).

Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.

A cross-linked complex between horse pancreatic lipase and colipase.

The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.

Human milk bile-salt stimulated lipase: further investigations on the amino-acids residues involved in the catalytic site.

The bile-salt-stimulated lipase purified from human skim milk was modified with diisopropyl phosphofluoridate (DFP), N-ethyl-5-phenylisoxazolium-3'-sulfonate and ethoxyformic anhydride. These chemical modifications lead to the following results: (1) the inhibition of the enzyme by DFP is due to the phosphorylation of a single residue, probably a serine residue, which may represent the acylable group of the enzyme; (2) carbethoxylation of histidine residues leads to inhibition of the enzyme activity. Among the nine modified histidine residues, only one is essential for enzyme activity; (3) a free carboxyl group with a pKa of 5.4 is also involved in catalysis. These three essential residues are involved in the enzymatic hydrolysis of substrates whatever their physical state (soluble or emulsified). Upon treatment with DFP as well as with ethoxyformic anhydride, the enzyme remains able to bind to the model interface formed by siliconized glass-beads with almost the same efficiency (Kd between 4.1 and 7.4.10(-8) M) than the native bile-salt-stimulated lipase (Kd = 6.3.10(-8) M). Moreover, the modified and native enzymes occupy the same interfacial area (4000-4600 A2/molecule). By contrast, the enzyme modified by N-ethyl-5-phenylisoxazolium-3'-sulfonate reagent presents an interfacial area close to that of a denatured protein of size (approximately 18,300 A2/molecule) and a Kd more than 20-fold higher than that of the native enzyme. Solvent isotope effects measured on kcat/Km and kcat indicate that the catalytic mechanism of bile-salt-stimulated lipase involves transition states that are stabilized by hydrogen bonds as described in the two-step acylation-deacylation mechanism of serine enzymes.

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase.

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).