Monitoring of aglycons of yew glycosides (3,5-dimethoxyphenol, myrtenol and 1-octen-3-ol) as first indicator of yew presence.

The toxicity of yew (Taxus spp) is well known from ancient times and is mainly due to taxins acting as inhibitors of calcium and sodium transport across the cell membrane of cardiac myocytes. The confirmation of yew taxins in body fluids can be carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, before selecting this precise but expensive technique, an orientation test should be done to ascertain yew presence as toxic agent in the organism. As the 3,5-dimethoxyphenol (3,5-DMP), myrtenol and 1-octen-3-ol appear as glycosidically bound volatile compounds and are very yew specific, the detection of 3,5-DMP and the measurement of 1-octen-3-ol / myrtenol concentration ratio constitute reliable indicators of yew presence in forensic cases. The detection of these compounds is easily performed by gas chromatography-mass spectrometry (GC-MS) (SIM) after an enzymatic hydrolysis (β-glucosidase) allowing the release of volatile compounds from yew glycosides. Copyright © 2012 John Wiley & Sons, Ltd.

Rapid sample pre-treatment prior to GC-MS and GC-MS/MS urinary toxicological screening.

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC-MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200 μL of urine in less than 30 min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5 min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC-MS run to 10 min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC-MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC-COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

Diffantom: Whole-Brain Diffusion MRI Phantoms Derived from Real Datasets of the Human Connectome Project.

Food allergies are believed to be on the rise and currently management relies on the avoidance of the food. Hen's egg allergy is after cow's milk allergy the most common food allergy; eggs are used in many food products and thus difficult to avoid. A technological process using a combination of enzymatic hydrolysis and heat treatment was designed to produce modified hen's egg with reduced allergenic potential. Biochemical (SDS-PAGE, Size exclusion chromatography and LC-MS/MS) and immunological (ELISA, immunoblot, RBL-assays, animal model) analysis showed a clear decrease in intact proteins as well as a strong decrease of allergenicity. In a clinical study, 22 of the 24 patients with a confirmed egg allergy who underwent a double blind food challenge with the hydrolysed egg remained completely free of symptoms. Hydrolysed egg products may be beneficial as low allergenic foods for egg allergic patients to extent their diet. This article is protected by copyright. All rights reserved.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

Distribution profile of zwitterions and hydrolysis of esters derived from N-benzylpiperazine

Résumé Les esters sont des agents thérapeutiques largement utilisés comme médicaments et prodrogues. Leurs dégradation est chimique et enzymatique. Le Chapitre IV de cette thèse a comme objet l'hydrolyse chimique de plusieurs dérivés esters du 2,3-dimethoxyphenol. Des composés modèles ont été synthétisés dans le but de déterminer leur mécanismes de dégradation. Les profils d'ionisation et d'hydrolyse de ces composés ont permis d'identifier la présence d'une catalyse intramoléculaire basique par un atome d'azote non-protoné. Les effets électroniques exercés par les groupes phenylethenyle et phenylcyclopropyle influencent également la vitesse d'hydrolyse des esters. La résolution des problèmes liés à l'adsorption et la perméation est devenue à nos jours l'étape limitante dans la conception de nouveaux médicaments car de trop nombreux candidats prometteurs ont échoué à cause d'une mauvaise biodisponibilité. La lipophilie décrit le partage d'un médicament entre une membrane lipidique et son environnement physiologique aqueux, et de ce fait elle influence sa pharmacocinétique. Des études récents ont mis en évidence l'importance de la détermination de la lipophilie des espèces ionisées vu leur considérable impact biologique. Le Chapitre V de cette thèse est centré sur une classe particulière de composés ionisables, les zwitterions. Plusieurs methoxybenzylpiperazines de nature zwitterionique ont été étudiées. Leurs profils d'ionisation ont montré que dans un large intervalle de pH, l'espèce prédominante est le zwitterion. Les profils de lipophilie ont montré que leur lipophilie est plus élevée que celles des zwitterions courants. Une interaction électrostatique entre l'oxygène du carboxylate et l'azote protoné est responsable de ce profil et rend la plupart des zwitterions non-donneurs de liaison hydrogène. Ces deux aspects peuvent favoriser le passage de la barrière hémato-éncephalique. Les données biologiques ont par la suite confirmé cette hypothèse pour un certain nombre de composés. Résumé large public Les esters sont des composés souvent rencontrés en chimie thérapeutique. Ils sont dégradés en milieu aqueux par une réaction d'hydrolyse, avec ou sans la participation d'enzymes. Dans ce travail de thèse, une série d'esters ont été étudiés dans le but d'établir une relation entre leur structure et les mécanismes responsables de leur dégradation chimique. Il a été prouvé que la dégradation est accélérée par un atome d'azote non-protoné. D'autres mécanismes peuvent intervenir en fonction du pH du milieu. La présence d'une liaison simple ou double ou d'un groupe phenylcyclopropyle peut également influencer la vitesse de dégradation. Il est essentiel, dans la conception de nouveaux médicaments, d'optimiser les étapes qui influencent leur distribution dans le corps. Ce dernier peut être visualisé comme une série infinie de compartiments aqueux séparés par des membranes lipidiques. La lipophilie est une propriété moléculaire importante qui décrit le passage des barrières rencontrées par les médicaments. Des études récentes ont mis en évidence l'importance de déterminer la lipophilie des espèces ionisées vu leur considérable impact biologique. Dans ce travail de thèse a été étudiée une série particulière de composés ionisables , les zwitterions. Une relation a été établie entre leur structure et leur proprietés physico-chimiques. Une lipophilie plus élevée par rapport à celle des zwitterions courants a été trouvée. Une interaction entre les groupes chargés des zwitterions étudiés est responsable de ce comportement inattendu et rend la plupart d'entre eux non-donneurs de liaison hydrogène. Ces deux facteurs peuvent favoriser la pénétration cérébrale. Les données biologiques ont confirmé cette hypothèse pour un certain nombre de composés. Summary Esters are often encountered in medicinal chemistry. Their hydrolysis may be chemical as well as enzymatic. Chapter IV of this manuscript provides a mechanistic insight into the chemical hydrolysis of a particular series of basic esters derived from 2,3-dimethoxyphenol. Their ionization and pH-rate profiles allowed to identify the presence of an intramolecular base catalysis by a non-protonated nitrogen atom. Electronic effects exerted by the phenylethenyl and phenylcyclopropyl groups that are present in the structure of the esters also influenced their rate of hydrolysis. Numerous works in the literature witness of the importance of lipophilicity in determining the fate of a drug. Most published partition coefficients are those of neutral species. In contrast, no exhaustive treatment of the lipophilicity of charged molecules is available at present, and a lack of information characterizes in particular zwitterions. Chapter V of this manuscript provides an insight into the physicochemical parameters of a series of zwitterionic methoxybenzylpiperazines. Their ionization profiles showed that they exist predominantly in the zwitterionic form in a broad pH-range. An electrostatic interaction between the oxygen of the carboxylate and the protonated nitrogen atom is increases the lipophilicity of the investigated zwitterions, and prevents the majority of them to express their hydrogen-bonding capacity. These two aspects may favor the crossing of the blood-brain barrier. The available ratios PSt/PSf measured in vitro have confirmed this point for a number of compounds.

Energetics of RecA-mediated recombination reactions. Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle.

We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis. The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used. We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions. The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction. This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction.

Development of a method to investigate the hydrolysis of xenobiotic esters by a Mycobacterium smegmatis homogenate.

One of the main problems in combating tuberculosis is caused by a poor penetration of drugs into the mycobacterial cells. A prodrug approach via activation inside mycobacterial cells is a possible strategy to overcome this hurdle and achieve efficient drug uptake. Esters are attractive candidates for such a strategy and we and others communicated previously the activity of esters of weak organic acids against mycobacteria. However very little is known about ester hydrolysis by mycobacteria and no biological model is available to study the activation of prodrugs by these microorganisms. To begin filling this gap, we have embarked in a project to develop an in vitro method to study prodrug activation by mycobacteria using Mycobacterium smegmatis homogenates. Model ester substrates were ethyl nicotinate and ethyl benzoate whose hydrolysis was monitored and characterized kinetically. Our studies showed that in M. smegmatis most esterase activity is associated with the soluble fraction (cytosol) and is preserved by storage at 5°C or at room temperature for one hour, or by storage at -80°C up to one year. In the range of homogenate concentrations studied (5-80% in buffer), k(obs) varied linearly with homogenate concentration for both substrates. We also found that the homogenates showed Michaelis-Menten kinetics behavior with both prodrugs. Since ethyl benzoate is a good substrate for the mycobacterial esterases, this compound can be used to standardize the esterasic activity of homogenates, allowing results of incubations of prodrugs with homogenates from different batches to be readily compared.

Calpain hydrolysis of alpha- and beta2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration.

Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the alpha- and beta2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent alpha- and beta2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal alpha- and beta2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the beta2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated beta2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of alpha- and beta2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.

Enzymatic activity, ultrastructure and function in ganglia infected with a neurotropic virus.

This chapter attempts to answer the questions, how do the viruses reach the neurons, what are the alterations that they impose on the neuronal machinery, and what are the consequences of these alterations on the function of the infected neurons? The virus used for this research was the pseudorabies. Pseudorabies virus is transported from the eye to the superior cervical ganglion by retrograde axonal flow. In the sympathetic neurons, the virus induces an increased protein synthesis and tyrosine 3-monooxygenase activity, a transsynaptic increased activity of the cholineacetyltransferase and a great rise in the acetylcholine content. The virus also causes an abnormal spontaneous electrophysiological activity, which also seems to be of presynaptic origin, despite the fact that the virus never crossed the synaptic cleft.

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by 13C turnover from hyperpolarized [1-13C]acetate to [1-13C]acetylcarnitine

BACKGROUND: Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle. METHODS: Hyperpolarized [1-(13)C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The (13)C magnetic resonance signals of [1-(13)C]acetate and [1-(13)C]acetylcarnitine were recorded in vivo for 1min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios. RESULTS: Although separated by two biochemical transformations, a kinetic analysis of the (13)C label flow from [1-(13)C]acetate to [1-(13)C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were KM=0.35±0.13mM and Vmax=0.199±0.031μmol/g/min. CONCLUSIONS: The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results. GENERAL SIGNIFICANCE: This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.

A new factor stimulating peptidoglycan hydrolysis to separate daughter cells in Caulobacter crescentus.

Cell division in Gram-negative bacteria involves the co-ordinated invagination of the three cell envelope layers to form two new daughter cell poles. This complex process starts with the polymerization of the tubulin-like protein FtsZ into a Z-ring at mid-cell, which drives cytokinesis and recruits numerous other proteins to the division site. These proteins are involved in Z-ring constriction, inner- and outer-membrane invagination, peptidoglycan remodelling and daughter cell separation. Three papers in this issue of Molecular Microbiology, from the teams of Lucy Shapiro, Martin Thanbichler and Christine Jacobs-Wagner, describe a novel protein, called DipM for Division Involved Protein with LysM domains, that is required for cell division in Caulobacter crescentus. DipM localizes to the mid-cell during cell division, where it is necessary for the hydrolysis of the septal peptidoglycan to remodel the cell wall. Loss of DipM results in severe defects in cell envelope constriction, which is deleterious under fast-growth conditions. State-of-the-art microscopy experiments reveal that the peptidoglycan is thicker and that the cell wall is incorrectly organized in DipM-depleted cells compared with wild-type cells, demonstrating that DipM is essential for reorganizing the cell wall at the division site, for envelope invagination and cell separation in Caulobacter.

Epidermal growth factor (EGF) stimulation of cultured brain cells. I. Enhancement of the developmental increase in glial enzymatic activity.

Serum-free aggregating cell cultures of fetal rat telencephalon grown in the presence of 3 ng/ml (5 X 10(-10) M) epidermal growth factor (EGF) until day 12 showed 2- to 3-fold increased activities in the two glial enzymes, glutamine synthetase (GLU-S) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This effect was concentration-dependent, with maximal stimulation in cultures treated daily with 3 ng/ml EGF. Addition of EGF during the first 10 culture days was sufficient to produce a maximal stimulation of both GLU-S and CNPase on day 19, whereas treatments starting on day 12 were ineffective. The stimulation of GLU-S preceded that of CNPase. The EGF-induced increase in GLU-S activity was not directly dependent on the presence of insulin, triiodothyronine, or hydrocortisone in the medium, whereas insulin was required for the stimulation of CNPase. A single dose of 5 ng/ml EGF on day 2 caused a slight but significant decrease in DNA synthesis after day 6. The present results indicate that in serum-free aggregating cell cultures of fetal rat telencephalon EGF partially inhibits DNA synthesis, and stimulates an early step in glial differentiation.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFalpha levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Adapter and enzymatic functions of proteases in T-cell activation.

Proteases control many vital aspects of humoral and cellular immune responses, including the maturation of cytokines and the killing of target cells. Recently, it has become evident that triggering of the T-cell receptor controls T-cell proliferation through proteases such as mucosa-associated lymphoid tissue 1 (MALT1) and Caspase-8 that act both as adapters and enzymes. Here, we discuss the role of these and other proteases that are relevant to the control of the T-cell response and represent interesting targets of therapeutic immunomodulation.

A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin.

BACKGROUND: The activity of the renin-angiotensin system is usually evaluated as plasma renin activity (PRA, ngAI/ml per h) but the reproducibility of this enzymatic assay is notoriously scarce. We compared the inter and intralaboratory reproducibilities of PRA with those of a new automated chemiluminescent assay, which allows the direct quantification of immunoreactive renin [chemiluminescent immunoreactive renin (CLIR), microU/ml]. METHODS: Aliquots from six pool plasmas of patients with very low to very high PRA levels were measured in 12 centres with both the enzymatic and the direct assays. The same methods were applied to three control plasma preparations with known renin content. RESULTS: In pool plasmas, mean PRA values ranged from 0.14 +/- 0.08 to 18.9 +/- 4.1 ngAI/ml per h, whereas those of CLIR ranged from 4.2 +/- 1.7 to 436 +/- 47 microU/ml. In control plasmas, mean values of PRA and of CLIR were always within the expected range. Overall, there was a significant correlation between the two methods (r = 0.73, P < 0.01). Similar correlations were found in plasmas subdivided in those with low, intermediate and high PRA. However, the coefficients of variation among laboratories found for PRA were always higher than those of CLIR, ranging from 59.4 to 17.1% for PRA, and from 41.0 to 10.7% for CLIR (P < 0.01). Also, the mean intralaboratory variability was higher for PRA than for CLIR, being respectively, 8.5 and 4.5% (P < 0.01). CONCLUSION: The measurement of renin with the chemiluminescent method is a reliable alternative to PRA, having the advantage of a superior inter and intralaboratory reproducibility.