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Regular or thermally treated egg white powder or soy protein isolate were included in the recipes of gluten-free (GF) breads based on rice flour (RF) or a mixture of potato and cassava starches (CS-PS) to improve bread texture and structure. Electrical resistance oven heating was combined with in-line measurements of batter height, viscosity and carbon dioxide release during fermentation and baking. Crumb firming during storage (6 days) of the different breads was studied by texture, moisture content, amylopectin (AP) crystal melting and proton nuclear magnetic resonance analyses. Drop shape tensiometry showed that both egg white types are highly surface active. Their use resulted in high gas cell stability and ultimately in high volume breads with fine crumb structure. Batters containing soy protein isolate, which had a lower surface-activity, had low stability. This resulted in low volume breads with inhomogeneous crumb structure. During the first 24 h of storage, the crumb firmness increase was mainly caused by AP retrogradation and later on by crumb-to-crust moisture migration. Crumb firming was less pronounced in CS-PS than in RF breads. Egg white decreased the extent of crumb firming while soy protein increased it. Most notably, differences in crumb firming between samples were largely related to differences in initial bread specific volume and crumb structure rather than to differences in AP retrogradation or moisture migration. Thus, inclusion of ingredients which improve bread volume and crumb structure in GF bread recipes may also strongly improve the shelf-life of such breads. ispartof: FOOD HYDROCOLLOIDS vol:95 pages:406-417 status: Published online

An approach to identify β-secretase 1 (BACE1) fragment binders that do not interact with the catalytic aspartate dyad is presented. A ThermoFluor (thermal shift) and a fluorescence resonance energy transfer enzymatic screen on the soluble domain of BACE1, together with a surface plasmon resonance (SPR) screen on the soluble domain of BACE1 and a mutant of one catalytic Asp (D32N), were run in parallel. Fragments that were active in at least two of these assays were further confirmed using one-dimensional NMR (WaterLOGSY) and SPR binding competition studies with peptidic inhibitor OM99-2. Protein-observed NMR (two-dimensional 15N heteronuclear single-quantum coherence spectroscopy) and crystallographic studies with the soluble domain of BACE1 identified a unique and novel binding mode for compound 12, a fragment that still occupies the active site while not making any interactions with catalytic Asps. This novel approach of combining orthogonal fragment screening techniques, for both wild-type and mutant enzymes, as well as binding competition studies could be generalized to other targets to overcome undesired interaction motifs and as a hit-generation approach in highly constrained intellectual property space.

When forming amylose-lipid (AM-L) inclusion complexes, surfactants retard bread crumb firming. Some wheat endogenous lipids have structures similar to those of surfactants. Lipase use in bread making increases the level of free fatty acids and ‘lyso’ lipids which can form AM-L complexes. We here used three lipases (Lipopan F, Lecitase Ultra, and Lipolase) with different specificities and the surfactant sodium stearoyl lactylate (SSL) for studying the role of lipids in bread crumb firmness and storage induced crumb firming. The lipases and SSL similarly impacted bread crumb texture. Their use induced less pronounced crumb firmness and stiffness increases as well as a less pronounced decrease in resilience than in control bread loaves. Amylopectin (AP) retrogradation was slower but in the end proceeded to a similar extent, as noted with low-field nuclear magnetic resonance. Differences in AP retrogradation after 7 days of storage (as observed with Lipolase) were attributed to the location and type of lipids hydrolysed by the respective lipase enzymes. Lipase hydrolysis products originating from lipids in the free lipid fraction probably had more impact on AP retrogradation than the free fatty acids and ‘lyso’ lipids obtained by hydrolysis of lipids ‘bound’ to the gluten network.

Research efforts on gluten-free bread making have rapidly increased during the last decade. A lot of different approaches are being used to improve the quality of these products. The techniques used in gluten-free bread making research vary widely. This review focuses on the methodological aspects of gluten-free bread making research and extracts relevant data from all Web of Science peer reviewed research articles on gluten-free bread published from 2010 to date. Recipes and methodologies are grouped by (main) starch source and list other ingredients, additives and treatments used. The focus lies on the experimental setups typically used to analyze batter/dough and end product. Small deformation rheological measurements are typically performed on gluten-free batter/dough, along with several other batter/dough properties, but there is no clear link between these characteristics and the bread quality which typically is determined by volume and texture analysis or sensory evaluation. Some more recent techniques that have already been used on wheat bread or other bakery products are discussed as well. Their application in gluten-free bread making research may help extend the current knowledge. ispartof: Journal of Cereal Science vol:67 pages:92-111 status: published

Artemin (ARTN) is a neurotrophic growth factor of the GDNF ligand family that signals through the specific GFRα-3 coreceptor/cRet tyrosine kinase-mediated signaling cascade. Its expression and signaling action in adults are restricted to nociceptive sensory neurons in the dorsal root ganglia. Consequently, Artemin supports survival and growth of sensory neurons and has been studied as a possible treatment for neuropathic pain. We have developed a robust and sensitive cellular assay to measure ARTN biological activity. Using recombinant Artemin produced in Escherichia coli bacteria together with this specific assay, we demonstrate that ARTN is an exceptionally stable polypeptide. Multiple freeze-thaw cycles, incubation at elevated temperatures (up to 90 °C) for 0.5 h, prolonged storage at 4 °C, and exposure to conditions of different pH, salt concentration, and additives had no measurable effect on the biological activity of ARTN. In some of the tested conditions, partial removal of nine NH(2)-terminal amino acids of the ARTN protein occurred, but this truncation had no important effect on the ARTN signaling response. Consequently, we postulate that formulation and storage for in vivo testing of ARTN in neuropathic pain paradigms in animals and humans should be straightforward.

International audience; Along the Sopoti section of Mali Gjere mountain are found two distinct mid-Cretaceous marly-shaly-siliceous intervals that occur near the top of the Vigla Limestone Formation (Aptian-Albian) of the Ionian zone in southern Albania. The lower interval is relatively rich in carbonate content (36% CaCO3 on average) and radiolaria (10% on average). It contains black shale levels rich in organic matter (up to 8.5% total organic carbon (TOC)) of marine origin, which did not experience any strong thermal maturation as suggested by their very low Tmax values. The age of the lower interval is latest Barremian to Early Aptian based on its calcareous nannofossil and radiolarian assemblages. It is therefore regarded as the equivalent of the Fourcade Level of Greece, reflecting the Oceanic Anoxic Event 1a (OAE1a). The upper interval is richer in both carbonate content (60% CaCO3 on average) and radiolaria (20% on average) but it is practically devoid of any preserved organic matter. Its age straddles the Aptian-Albian boundary based on integrated biochronologic data of dinoflagellates, calcareous nannofossils and radiolaria. It is tentatively considered as the sedimentary expression of OAE1b (sensu Leckie et al., 2002) in the Ionian zone of Albania. The presence of large Assipetra nannoliths in both shaly-siliceous intervals and the relative abundance of radiolaria suggest that their accumulation took place during periods of higher productivity in the Ionian zone of Albania. = Deux horizons argilomarneux bien distincts sont identifié s vers le sommet de la Formation crétacée des Calcaires de Vigla de la zone ionienne. Ils sont étudiés en détail le long de la coupe de Sopoti de l'Albanie méridionale. L'horizon inférieur est relativement riche en carbonates (36% de CaCO3 en moyenne) et radiolaires (10% en moyenne). Il contient de nombreux niveaux riches en matière organique d'origine marine (jusqu'à 8,5% de COT). Des valeurs très faibles de Tmax indiquent que la matière organique n'a pas subi une forte maturation thermique. Sur la base des nannofossiles calcaires et des radiolaires l'âge de cet horizon inférieur est corrélé avec le Barré mien terminal - Aptien inférieur et par conséquent considéré comme équivalent du niveau Fourcade (OAE1a) de Grèce. L'horizon supé rieur est plus riche en carbonates (60%de CaCO3 en moyenne) et radiolaires (20% en moyenne), mais il est pratiquement dé pourvu de matière organique. Son âge couvre la limite Aptien-Albien sur la base des dinoflagellés, des nannofossiles calcaires et des radiolaires et il est provisoirement corrélé avec l'OAE1b. L'abondance relative des radiolaires, la dominance de Radiolaires Nassellaires multicyrtides et la présence de larges nannolithes Assipetra au sein de ces deux horizons suggèrent que leur accumulation a eu lieu durant des intervalles d'eutrophisation et d'une production biosiliceuse accrue des radiolaires au sein de la zone ionienne.

International audience; The performances of all solid state potassium selective electrodes with polypyrrole film as solid internal contact were evaluated. Potentiometric measurements revealed near Nernstian responses close to values already given in the literature for classic ISEs. In addition, ac-electrogravimetry was used in order to study the ions and solvent motions at the membrane/electrolyte interface. We have found that potassium ions enter the selective membrane firstly but solvent molecules and anions are also participating in the phenomenon.

The impedance of an ion-selective electrode (ISE) to potassium with valinomycin as the ionophore and KBΦ4 as the ionic sites was measured. The membrane was made of PVC with DNP as a plasticizer. The influence of the content of the plasticizer and the membrane thickness was investigated. The concentrations of the ionophore and the ionic sites were changed to modify the response on the ISE. Finally, the selectivity was tested in terms of impedance. All impedance experiments were correlated to the potentiometric curves plotted under the same experimental conditions. The measured impedances were compared to the model proposed in the previous paper (paper I). A good agreement was found.

Models of an ion selective electrode involving an ionophore and mobile sites in a membrane are proposed. The first model, called the phase boundary potential model, supposed thermodynamic equilibrium; it allows the concentrations of the various species to be calculated. Then, a kinetic model, which takes into account the ionic transfer at the membrane|solution interfaces, was derived. The impedance of the membrane was calculated. It shows that a membrane with nernstian behavior shows only one capacitive loop in the impedance diagram, which is related to the conductivity and dielectric properties of the material of the membrane. Non-nernstian behavior is related to slow ionic transfer at the membrane|solution interfaces or/and transport limitation of the species in the membrane. Finite rate constants of the ionic transfer lead to a capacitive loop in the middle frequency range, whereas finite rate transport leads to a diffusional impedance in the low frequency range.

Human β-secretase (BACE-1) is a type I integral membrane aspartic protease that catalyzes the internal cleavage of the amyloid precursor protein (APP), generating the N-terminus of the Aβ peptide. The generation and subsequent extracellular deposition of Aβ 1−42 peptide into amyloid plaques in the brain constitute one of the hallmarks of Alzheimer’s disease (AD), a common debilitating neurodegenerative disorder. Inhibition of BACE-1 is considered an excellent therapeutic strategy against AD. To generate pure enzyme for protein crystallography and subsequent structure-based drug design, we have expressed a soluble, unglycosylated, 6×His-tagged form of proBACE-1 in insect cells using baculovirus infection. To avoid production of a mixture of the pro-enzyme form and the mature form of BACE-1, the proprotein convertase furin was coexpressed with proBACE-1, leading to almost complete proteolytic activation of the recombinant enzyme. The mature enzyme was secreted in the conditioned medium of BACE-1/furin coinfected HighFive insect cells. Secreted BACE-1 protein was purified to homogeneity from the medium using subsequent Ni-chelate affinity chromatography, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. To avoid autoproteolysis, all purification steps were performed at pH values outside the activity range of BACE-1. The purified, biologically active enzyme was homogeneous on SDS/PAGE and had the expected sequence and molecular mass determined by N-terminal amino acid sequencing and mass spectrometry, respectively. Moreover, the preparation showed a single peak of the expected size with only 17% polydispersity using dynamic light scattering analysis. The yield of BACE-1 from fermentation cultures was approximately 0.1 mg pure enzyme per liter of cell culture medium. The purified protein was successfully used to generate BACE-1/inhibitor co-crystals and to determine the crystal structure of the complex by X-ray analysis. The availability of substantial quantities of active, homogeneous enzyme will be of great help in future structure-based drug design efforts in the search for efficient protease inhibitor drugs to treat AD.

Cyclic polyisoprenes have been synthesized by reaction of α,ω-dilithiopolyisoprenes with dichlorodimethylsilane or 1,2-bis(isopropenyl-4-phenyl) ethane (BIPE). Using the dihalide compound, the effect of the solvent polarity on the coupling reaction is more pronounced in the case of α,ω-dilithiopolyisoprene than with monofunctional polyisoprenyllithium. The yield in cyclic compound falls from 88% in pure hexane down to 53% in the presence of 15 vol% of tetrahydrofurane (THF). Using the nonconjugated diene (BIPE) as linking agent the addition of THF is required but the formed cycle retains its living character and allows the synthesis of cycles having two arms (after addition of isoprene) and of a bicyclic structure after a second cyclization reaction.

Lipopan F and Lecitase Ultra lipases were used in straight dough bread making to study how wheat lipids affect bread loaf volume (LV) and crumb structure setting. Lipase effects on LV were dose and dough piece weight dependent. The bread quality improving mechanisms exerted by endogenous lipids were studied in terms of gluten network strengthening, which indirectly stabilizes gas cells, and in terms of direct interfacial gas cell stabilization. Unlike diacetyl tartaric esters of mono- and diacylglycerols (DATEM, used as control), lipase use did not impact dough extensibility. The effect on dough extensibility was therefore related to its lipid composition at the start of mixing. Both lipases and DATEM strongly increase the levels of polar lipids in dough liquor and their availability for and potential accumulation at gas cell interfaces. Lipases form lysolipids that emulsify other lipids. We speculate that DATEM competes with (endogenous) polar lipids for interacting with gluten proteins.

Cationic polymerization of hexamethylcyclotrisiloxane (D3) initiated by HCl and antimony pentachloride (SbCl5) may give a high polymer (HP) with controlled molecular weight in high yield, and only small amounts of cyclic compounds. Theoretical Mn's were formerly obtained with D3 using as initiator either triflic acid or a combination of a silyltriflate activated by triflic acid. However, considerable amounts of cyclic compounds were also formed, e.g. 50 wt % D6/HP and up to 20% macrocycles. D6 was mainly formed by a reaction involving transitory oxonium ions and macrocycles by cyclization of silanol esters. Using two other initiating systems, the formation of D6 and macrocycles was significantly decreased and was sometimes suppressed. The polymerization was rapid with SbCl5 and HCl at −10 °C, producing about 90% high polymer of Mn ≥ 105, 10−12% small cycles (only 3% D6) and no macrocycles. The Mn's agreed with an initiation by HCl and a propagation involving the silanol end groups, which explains the abse...

The synthesis of heterotelechelic polystyrene chains containing α-hydroxy-ω-carboxy end groups and their intramolecular cyclization are described. The controlled free radical polymerization of styrene was carried out using 4,4‘-azobis(4-cyanovaleric acid) as the initiator and 4-hydroxy-TEMPO as the terminator, to generate difunctional macromolecules with molar masses in the range 1−10 kg mol-1. The cyclization reaction is clearly evidenced by infrared spectrometry and size exclusion chromatography (SEC). In addition, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and liquid chromatography at the exclusion−adsorption transition point (LC PEAT) analyses were successfully applied. The yield of low molar mass (1 kg mol-1) macrocycles was close to 95%. For higher molar masses, polycondensate byproducts and nonfunctionalized chains due to styrene thermal initiation reduce the cyclization efficiency.

Macrocyclic polystyrenes were prepared by coupling a two-ended living precursor dianions with 1,3-bis(1-phenylethylenyl)benzene (DDPE). Experiments were performed in a drybox apparatus, and macrocy...

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors α (GFRα), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of125I-GDNF from GFRα1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low 125I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1–20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a KD of 11 pm for 125I-GDNF is detected only when GFRα1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRα1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.

Gelatinase B is a matrix metalloproteinase (MMP-9) involved in tissue remodeling, development, cancer, and inflammation. Neutrophils produce three major forms of (pro)gelatinase B: 92 kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (NGAL). In contrast to the case for other proteinases, little information about the glycosylation of any natural human MMP is available. Here, both gelatinase B and NGAL were purified from human peripheral blood neutrophils, and the entire contents of the released N- and O-glycan pools were analyzed simultaneously using recently developed high-performance liquid chromatography-based technology. The results are discussed within the context of the domain structure of gelatinase B and a molecular model of NGAL based on data from this study and the three-dimensional nuclear magnetic resonance (NMR) structure of the protein. More than 95% of the N-linked glycans attached to both gelatinase B and NGAL were partially sialylated, core-fucosylated biantennary structures with and without outer arm fucose. The O-linked glycans, which were estimated to comprise approximately 85% of the total sugars on gelatinase B, mainly consisted of type 2 cores with Galbeta1,4GlcNAc (lactosamine) extensions, with or without sialic acid or outer arm fucose. This paper also contains the first report of O-linked glycans attached to NGAL. Although both proteins were isolated from neutrophils and contained O-linked glycans mainly with type 2 cores, the glycans attached to individual serine/threonine residue(s) in NGAL were significantly smaller than those on gelatinase B. In contrast to NGAL, gelatinase B contains a region rich in Ser, Thr, and Pro typical of O-glycosylated mucin-like domains.

The synthesis of poly(methylhydrogeno)siloxane (PMHS) by cationic polymerization of 1,3,5,7-tetramethylcyclotetrasiloxane (D4H) was studied in aqueous emulsion using dodecylbenzenesulphonic acid (DBSA) as emulsifier/initiator. With pure DBSA, the rates of polymerization are very high and the PMHS formation is not well-controlled (cross-linking reactions, emulsion not very stable). The addition of the sodium salt of DBSA as coemulsifier does not give satisfactory results. In the presence of a neutral coemulsifier (Brij35), the process leads to the formation of linear PMHS of controlled molecular weight in the range 7000–70 000 g mol −1 with a yield of 90%. No cross-linking reaction was observed, showing the good stability of the SiH bond in these experimental conditions. Preliminary investigations lead to the conclusion that the polymer is probably formed by initiation, propagation and termination reactions taking place simultaneously at the surface of 200 nm diameter monomer particles. Nevertheless, the peculiar mechanism of these reactions seems more complicated than those previously found in the case of anionic polymerization of D4 in aqueous emulsion. © 1999 Society of Chemical Industry

A kinetic study of the anionic polymerization of octamethylcyclotetrasiloxane (D 4 ) in aqueous emulsion has been carried out in the presence of ionic additives. The rate of polymerization of several cyclosiloxanes has been compared, leading to additional evidence for an interfacial mechanism of polymerization. The emulsion process has been applied to the cationic polymerization of D 4 and of tetramethylcyclotetrasiloxane (D H 4 ) initiated by dodecylbenzenesulfonic acid. Very efficient for the synthesis of linear polymethylhydrogenosiloxanes (PMHS), these conditions did not seem suitable for the polymerization of D 4 . The extension of the process to other heterocyclic monomers is discussed through the anionic polymerization of phenylglycidylether.

The need for new optically active monomers and polymers is conducive to the setting up of stereospecific synthesis routes starting from chiral precursors. The biomass can be considered as a major source for extracting such biomolecules aimed at chemoenzymatic transformation and further polymerization. Due to its versatility, β-methylaspartate ammonia-lyase, from cell-free extracts of Clostridium tetanomorphum, has been used in the bioconversion of alkylfumarates into optically active pure 3-alkylaspartic acids with alkyl=methyl, ethyl, isopropyl. These amino acids have been transformed in several steps into optically active benzyl 3-alkylmalolactonates leading to semi-crystalline polyesters. 3-Methylaspartic acid includes two chiral centers and the racemic compound containing the four stereoisomers can be prepared by a multiple step synthesis. The ability of β-methylaspartase to catalyse both syn- and anti-elimination of ammonia from natural 3-methylaspartic acid has been expressed to retain one stereoisomer and this bioconversion is a preparative method for obtaining unnatural stereoisomers. Moreover, the catalytic hydrogenolysis of the benzyl α,β-substituted β-lactone yields stable 3-alkylmalolactonic acid which can be coupled with functional alcohols and copolymerized. At last the introduction of (2S)-3,3-dimethyl-2-butanol, using Rhodotorula glutinis as microorganism in a biological synthesis step, as chiral ester pendant group, has conducted to optically active polyesters with very high melting transition temperatures. The combination of bioconversion and chemical synthesis is a very useful tool for building hydrolyzable functionalized polyesters required for temporary applications.

The anionic polymerization and copolymerization of butyl and benzyl malolactonates initiated with potassium acetate/dibenzo-18-crown-6 or [222] complex have been studied at 40°C in bulk and in dichloromethane and tetrahydrofuran solutions. Kinetics and polymer molecular weights were followed by SEC. In bulk and for conversion below 30%, molecular weights are close to those calculated for a living process then the conversion increase leads to a significant discrepancy between theoretical and measured molecular weights. In solution, side reactions take place significantly and no evident relationship between molecular weight and initiator concentration can be established, even at low conversion. Same situation was observed in the case of block copolymers synthesis. At last, evidence for transfer reactions has been shown by using model reactions.

Transformation in bacteria is the uptake and incorporation of exogenous DNA into a cell's genome. Several species transform naturally during a regulated state defined as competence. Genetic elements in Streptococcus pneumoniae induced during transformation were identified by combining a genetic screen with genomic analysis. Six loci were discovered that composed a competence-induced regulon. These loci shared a consensus promoter sequence and encoded proteins, some of which were similar to proteins involved in DNA processing during transformation in other bacteria. Each locus was induced during competence and essential for genetic transformation.

The stereochemistry of the cycloaddition of nitroethylene, as a model of 6-nitrobenzofuroxans, with ethenol and vinylamine has been studied at the ab initio DFT level. MO analysis reveals that these inverse electron demand Diels−Alder reactions are under frontier orbital control. A one-step reaction pathway involving a dissymmetrical transition state is favored relative to a two-step mechanism in which the intermediate has essentially a diradical character. Study of reactions of a series of 4-substituted 6-nitrobenzofuroxans with various enol ethers and enamines at the semiempirical AM1 level shows that in these instances a stepwise mechanism involving a short-lived diradical intermediate is likely to occur. Relative rates and stereoselectivities of these reactions as a function of the nature of reactants is discussed.

The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.

Calculated (MP4/6-31G ∗∗ //MP2/6-31G ∗∗ and/or CASSCF/6-31G ∗∗ ) energies and equilibrium geometries of the lowest triplet and singlet states of phosphinidenes PAX 3 ( A = C , Si ; X = H , F ) and phosphaethenes XP = AX 2 are reported. As previously noted for PCH 3 , singlet phosphinidenes exhibit a slight C s distortion from C 3 v symmetry; the triplet-singlet separation is rather insensitive to the nature of the substituent. For phosphaethenes, of C s symmetry, the singlet-triplet separation reflects the strength of the π-bond and drops to 14.1 kcal mol −1 for FPSiF 2 . Singlet phosphaethene is the minimum on the corresponding PES, except for PSiF 3 , for which both triplet and singlet phosphinidenes are found below singlet phosphaethene. The transition states for the phosphinidene-phosphaethene transpositions have been determined as well as the singlet and triplet states. PSiH 3 is expected to undergo a fast transposition. For both compounds, fluorine substitution significantly raises the transposition barrier.

Earlier data had shown that monomer reactivities and polymerization rates of cyclodimethylsiloxanes D x ([Si(CH 3 ) 2 O] x ) increased in the order D 4 5 6 for polymerizations initiated by trifluoromethanesulfonic acid in methylene chloride. The larger reactivity of D 3 was assumed to result from a larger ring strain. No data were available for larger rings. In the present article, the rates of polymerization of D 5 , D 6 and D 7 were compared, the initial rates increasing in the order Ro D 7 = 3 Ro D 6 = 7 Ro D 5 . A copolymerization of D 6 and D 7 also gave R c o D 7 = 3 R c o D 6 . The increase in rate with ring size may result from an increase in the flexibility of the large rings permitting a ring to reach the transition state more easily. At the beginning of the homopolymerization of D 6 and D 7 , a kinetic enhancement in the cyclic dimer formation (D 12 or D 14 ) was also observed. This was attributed to the presence at the beginning of the polymerization of significant amounts of silanol esters HD 2 x OSO 2 CF 3 , which is in agreement with the formation of larger macrocycles during the same period.

The asymmetric lactone (3 S, 4 R)-3-methyl-4-benzyloxycarbonyl-2-oxetanone (6) was anionically polymerized to give an insoluble, crystalline, highly isotactic polymer with (2 S, 3 S)-benzyl β-3-methylmalate repeating units. Solubility was achieved by copolymerization of 6 with the recemic (R, S)-butyl malolactonate (7). The semicrystalline copolymer was characterized (Mn = 107 000, Tg = 29,6°C, Tm = 161°C, [α] = 1,5 deg · dm−1 · g−1 · cm3) and its stereosequence investigated by 13C NMR.

The production of gelatinase B by macrophages is relevant in the immunological and migratory functions of macrophages. CGP 41251, an inhibitor of protein kinase C (PKC), was found to stimulate the expression of gelatinase B in macrophages, as shown by the study of two different monocytic/macrophagic cell lines, mouse RAW 264.7 and human THP-1 cells. When human monocytis and rat peritoneal macrophages were treated with CGP 41251, insignificant increases of 10 and 25% were obtained. This can possibly be due to the presence of contaminating cells in these two enriched populations, since the CGP 41251 treatment of non-macrophagic cell lines inhibited their PMA-induced gelatinase B production. Taken together, these results suggest that the stimulatory effect of CGP 41251 is specific to cells of the monocytic lineage. Using RAW 264.7 cells as a model, the effect of CGP 41251 is additive to that obtained using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), as revealed by gelatin zymography and Northern blot analysis. The stimulatory effect of CGP 41251 on gelatinase B production in RAW 264.7 was: (a) inhibited by calphostin C (as is the LPS-induced response), indicating a PKC-dependence; (b) inhibited by dexamethasone (as opposed to the LPS-induced response); and (c) enhanced by addition of trans-retinoic acid (RA). In fact, RA can induce gelatinase B production, either alone or in synergy with LPS and/or CGP 41251, since the combination of the three agents gives the highest gelatinase B response, at both the protein and the mRNA levels. This represents an important observation considering that RA is now being tested as an anti-cancer agent and proposed for prevention studies.

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcbeta1-3Gal, GalNAcbeta1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo.

Human gelatinase B was produced from peripheral blood neutrophils and purified by affinity chromatography on gelatin sepharose. This material was used as an antigen to prepare mouse monoclonal antibodies (mAb). The resulting hybridomas were selected on the basis of binding to biotinylated antigen and by a sandwich ELISA using gelatinase-B-specific polyclonal rabbit antiserum and pure natural antigen. Five of these mAb were selected for further characterization. They all displayed variable epitope specificity, binding capacity and inhibitory activity. Whereas mAb REGA-2D9 and REGA-3G12 showed the strongest binding to biotinylated gelatinase B and natural gelatinase B, respectively, mAb REGA-2F9 did not bind biotinylated antigen. None of the mAb displayed cross-reactivity to gelatinase A in a direct ELISA. The mAb REGA-1G8 was found to cross-react with human serum albumin. The binding capacity of the other four mAb with leukocyte gelatinase B was compared and a sensitive sandwich ELISA was developed with the antibodies REGA-3G12 and REGA-2D9 (detection limit 0.5 ng/ml). The mAb REGA-3G12 was unique in that it inhibited catalysis by gelatinase B. This was shown by assaying the degradation of nasal septum type II gelatin in the presence and absence of each of the five mAb. Furthermore, mAb REGA-3G12 inhibited the degradation of biotinylated gelatin in a microtiterplate solution assay. In addition to the potential use of the inhibitory mAb REGA-3G12 in the treatment of diseases with excessive gelatinase B production, several of the described mAb are useful as diagnostic probes to detect gelatinase B in body fluids and tissue samples of patients with multiple sclerosis, rheumatoid arthritis and cancer.

Silyl trifluoromethanesulfonates are inactive for the polymerization of cyclosiloxanes in the complete absence of trifluoromethanesulfonic (triflic) acid, but polymerization occurs in its presence. Using a triflic ester as tagged silyl triflate (benzyldimethylsilyl triflate) the degree of polymerization (DP n ) of the linear polymer formed with hexamethylcyclotrisiloxane (D 3 ) in CH 2 Cl 2 at 20°C is inversely proportional to the ester concentration and near to the theoretical value. The polymer contains one PhCH 2 (CH 3 ) 2 Si group per macromolecule, in agreement with a propagation occurring on the monoester PhCH 2 (CH 3 ) 2 SiD 3x OTf. By comparison with experiments using the acid alone, polymerizations made in the presence of this ester lead to the suppression of the formation of macrocycles and to a strong decrease of that of D 9 and D 12 . But the amount of D 6 is only slightly reduced, which agrees with the hypothesis of its main direct formation on the active sites and not by ring closure of a silanol ester. The rate of D 3 polymerization is internally 1 st order in [D 3 ] and externally proportional to both acid and ester initial concentrations, in agreement with a propagation involving silyl triflates activated by the acid, interconverting with transitory siloxonium ions leading to D 6 formation by a ring expansion involving the polymer chain.

Pretreatment of LPS-induced RAW 264.7 cells with three PKC inhibitors suggests that induction of TNF-alpha, nitric oxide (NO), gelatinase B (Gel B) and IL-6 involves at least three distinct signalling pathways. We confirmed the PKC dependence of TNF-alpha and NO productions and found that Gel B was inhibited by Calphostin C (CAL), but potentiated by staurosporine (STAR) and CGP 41 251. IL-6 production was stimulated by the three inhibitors. Our results indicate that up-regulation of Gel B, TNF-alpha and NO seems to involve PKC at different levels, whereas up-regulation of IL-6 production appears to be PKC-independent. However, IL-6 production in RAW 264.7 cells seems to be down-regulated by a PKC-dependent feedback mechanism.

Polymerization of hexamethylcyclotrisiloxane (D3) initiated by trifluoromethanesulfonic acid (triflic acid TfOH) was carried out under vacuum, in methylene chloride solution at 20°C. The reaction rate is first order in [D3] and the constant active sites concentration [P*] varies as [P*] = k · [TfOH] · [D3]. The two main products of the reaction, both formed in similar amount from the beginning, are the dimer D6 and a high-molecular-weight polymer (HP). The other cyclic products also formed are the multiples of D3 (D9, D12, …), macrocycles MC (number-average molecular weight Mn ≈ 104) and small amounts of D4, D5 … (with [D5] > [D4]). The concentration of HP, MC, D6, D9, D12, … and the Mn of the high polymer grow proportionally to conversion. The main reaction giving the high polymer and D6 is interpreted as a chain reaction, the growing HP bearing at each end potentially reactive silyl triflates which may be activated by the residual acid. D6 is mainly formed by a special type of back-biting reaction involving transitory tertiary siloxonium ions. The other cycles D3x and the macrocycles mainly result from cyclization reactions of a second population of growing macromolecules which bear a silyl ester at one end and a nucleophilic silanol group at the other end.

To identify elements participating in the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistently transformed 10-fold less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease-like protein), which encodes a putative substrate-binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide-binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to Spo0KA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PlpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [3H]-palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the plpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were > 90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (iii) the plpA mutation decreases the expression of a competence-regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate-permease interaction modulates the process of transformation.

The first step of the growth of a metal particle supported on an oxide has been theoretically simulated using the extended Huckel molecular orbital (EHMO) approach. The nucleation site at the oxide surface is modeled by a M(OH)x complex (x=3 and 5) and its interaction with either an isolated metal atom or a dimer theoretically calculated. The latter can approach the nucleation site with its internuclear axis either perpendicular or parallel to the surface. The stabilization energy, E s , expressed as the energy difference between the nucleation site and the metal (mono- or dimer) moieties at infinite separation and at bonding distance (2.25 A), has been plotted as a function of the original number of electrons at the nucleation site

A general feature of the cationic polymerization of all cyclodimethylsiloxanes is the formation of various cyclic products (cyclics) together with that of a linear high polymer. However, the types of cyclics as well as their rate of formation may vary considerably according to the number x of D units ((CH3)2SiO units) in the monomer. The case of initiation by trifluoromethanesulfonic (triflic) acid in methylene chloride solution at 20°C has been particularly studied. With D4, D5, D6 and D7, for which the polymerization rate increases with the size of the ring, all types of cycles Dx are formed in relative amount decreasing with their size ([D7] < [D6] < [D5] < [D4]). The high polymer final concentration and molecular weight are independent from triflic acid concentration. This may result from a polymerization-depolymerization reaction, involving all the cyclics formed by back-biting reactions occurring with silyl triflates activated by the acid, and leading finally to an equilibrium. The situation with D3 is completely different. The high polymer (HP) and the cyclics (D3x multiples of D3 like D6, D9, …) are formed simultaneously under kinetic control. The yields of the various cyclics (formed in amount often larger than that of the HP) are proportional to that of the linear HP. The latter is formed from the beginning of the reaction with a molecular weight proportional to HP yield and inversely proportional to the acid concentration. The opposite role of added water on the polymerization is discussed: an activating effect for D3, and a desactivating one for D4, D5 and D6. “Copolymerization” experiments between D3 (or D4) and tetramethyldisiloxane diol confirmed the effect of water and gave new informations about the occurrence - or absence - of condensation reactions in the mechanism of the growth of the polymer chains. A discussion leads to the conclusion that while the cationic polymerization of D4 by triflic acid is propagated by activated triflic esters, that of D3 may also involve the monomer activated by the higher hydrates of the acid and linear oligomeric silanol esters. The latter, formed continuously, may also give the D3x cyclics by intramolecular heterocondensation.

The adenylate cyclase toxin (ACT) of Bordetella pertussis has been shown to penetrate eucaryotic cells and produce a rapid elevation in intracellular cAMP which leads to altered cell function. Recent studies have demonstrated an intracellular state for the bacteria within professional and non-professional phagocytes. A virulent strain was compared to two ACT defective strains to determine if this toxin contributes to intracellular survival within human macrophages. When challenged by 106 macrophages/ml in a cell invasion assay, 103 bacteria/ml were recovered from samples containing the ACT defective strains. These values were two log units less than the number of bacteria recovered from samples containing the isogenic parent. The binding and uptake of all strains by the macrophages were equivalent, suggesting that ACT does not affect adhesion nor endocytosis but rather protects against macrophage killing following uptake. Drug-induced elevation of cAMP levels within the macrophage by forskolin increased the number of surviving bacteria in samples containing the mutant strains to values equal to those obtained with the parent strain. Therefore, the protective effect conveyed by ACT is the result of toxin-induced elevation of cAMP within the macrophage concomitant with bacterial uptake.

Polymerization of 1,3,5,7 tetramethylcyclotetrasiloxane (D 4 H ) by trifluoromethanesulphonic acid in CH 2 Cl 2 solution at 20° has given high polymers (HP) with molecular weights > 10 5 . For sufficiently high monomer concentrations (e.g. 1.9 mol · l −1 ), the HP yield may reach 60%. Small cycles are formed together with the high polymer by back-biting reactions as in the case of octamethylcyclotetrasiloxane (D 4 ) but the polymerization rate is much higher than that of D 4 and near that of D 3 . The kinetics are quite different and much simpler than those observed for the dimethylcyclosiloxanes: the reaction is still internally 1st order in monomer, but the apparent rate constants are 1st order in both acid and monomer concentrations. This effect may result from the much larger reactivities of silyltrifluoromethanesulphonates derived from this monomer.

Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.

Cyclodimethylsiloxanes such as hexamethylcyclotrisiloxane (D3) and octamethyl-cyclotetrasiloxane (D4) have quite different reactivities and generally do not give the same types of cyclic products. An examination of analogies and differences between these two monomers is made in the case of initiation by trifluoromethane sulfonic acid, for both the formation of water during the reaction and the effect of water and other additives. The differences in the formation of water are much less than was expected and perhaps not really significant. A second analogy is that for both monomers, the retarding or inhibiting effect of water is much stronger when it is premixed with the acid rather than mixed in situ, this being attributed in this last case to a possible competition between monomer and water for the first reaction with acid. From the variation of the rates when the mole ratio [H2O]/[Acid] increases, it is concluded that for the initiation reaction water is a retarder or inhibitor for both monomers while for propagation it is an inhibitor for D4 but a very effective cocatalyst for D3. After a comparison of the effects of added water, silanol and triflic esters on propagation reaction and cyclics formation for these two monomers, and a discussion of the possible active centres, it is concluded that the mechanism of propagation should be different for D3.

A theoretical study of the enantioselective Michael-type addition of chiral secondary enamines to enones has been achieved. In a first step, the structures of various free enamines have been investigated at the ab initio and MNDO levels. The results clearly show that upon substitution of the prototype vinylamine, the N-center is pyramidalized. The study of enamines with chiral N-substituents such as (S)-Ph(Me)CH or (S)-cyclohexyl(Me)CH reveals a very complex pattern, where up to 8 local energy minimums are characterized whose examination shows that no prediction can be. done regarding the final enantioselectivity of their reaction with enones. These sets of conformers can be regarded as nearly energetically degenerate, at least for the three or four ones of lowest energy. The study of the compact complexes formed between the latter optimal conformers and acrylaldehyde shows that: (i)syn complexation with respect to the N lone pair is the only one which remains possible for steric reason, (ii) small geometrical rearrangements take place as the complexation proceeds, (iii) no clear-cut correspondence exists between the relative sequence of the low-energy conformers of the free enamines and the sequence of the low-energy complexes, (iv) the examination of the relative gradients of the complexation energies provides an index for predicting the relative facilities of the enantiometric pathways, in good agreement with the experimental facts. Our study emphasizes the great complexity of systems of realistic size and brings about critical conclusions regarding classical ad hoc models.

A theoretical study (SCF PS-HONDO + CI) of the stability of the tribromide anion Br3− has been carried out. On the basis of qualitative MO arguments and MNDO calculations, we propose a first approach to the mechanism of electrophilic aromatic substitution by Br3−. It involves an axial attack of linear Br3−, the most electrophilic sites of which are terminal bromine atoms, although they bear a negative charge. This model suggests some arguments to explain the para versus ortho selectivity experimentally observed. Calculations performed on a series of MBr3 systems emphasize the importance of the countercation for the rate of the reaction as well as for its selectivity. Keywords: bromination, tribromide, mechanism, parabromophenol.

A new aspartic protease inhibitory chemotype bearing a 2-amino-3,4-dihydroquinazoline ring was identified by high-throughput screening for the inhibition of BACE-1. X-ray crystallography revealed that the exocyclic amino group participated in a hydrogen bonding array with the two catalytic aspartic acids of BACE-1 (Asp(32), Asp(228)). BACE-1 inhibitory potency was increased (0.9 microM to 11 nM K(i)) by substitution into the unoccupied S(1)' pocket.