From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in Insects

One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects

Discrimination of diastereoisomers by electron ionization mass spectrometry in the 1,3-dioxane series

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Off-line capillary electrophoresis/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry for analysis of synthesized poly(amido)amine dendrimers.

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Direct TLC/MALDI–MS coupling for modified polyamidoamine dendrimers analyses

International audiencePolyamidoamine (PAMAM) are synthetic dendrimers which present attractive properties for the biolog- ical and biomedical fields, as they proved to be efficient drug and gene carriers. In order to increase their transfection efficiency, chemical modifications of the amino end-groups had been reported. In this work, the synthesis of the ammonia-cored G1(N) PAMAM and the consecutive chemical modification with glycine or phenylalanine amino-acids were monitored using the coupling of thin layer chromatography (TLC) with matrix–assisted laser desorption ionization–mass spectrometry (MALDI–MS). Thus, the moni- toring of the PAMAM synthesis included the identification of the by-products such as defective structures of PAMAM dendrimers as well as the study of phenylalanine-grafted PAMAM oligomer distribution

Glycine-modified polyamidoamine dendrimers: synthesis and structural characterization using nuclear magnetic resonance, ion-mobility mass spectrometry and capillary electrophoresis

We present here the preparation and the structural characterization of first-generation ammonia-cored polyamidoamine (PAMAM) dendrimers modified with glycine residues. Chemical modification of the dendrimer was done to increase the biocompatibility of these compounds, known to be effective delivery agents for drugs or genes. Fully modified PAMAM [Gly6G1(N)] on the one hand and partially modified [GlynG1(N), with n = 0 to 6)] on the other hand were obtained depending on the experimental conditions. The resulting modified PAMAM dendrimers have to be cautiously characterized to understand and interpret their physico-chemical and biochemical properties as well as to control their chemical design. The structural characterization was carried out using ion mobility spectrometry-mass spectrometry (IM-MS), multistage tandem mass spectrometry (MSn), accurate mass measurements by high resolution–mass spectrometry (HR-MS), two dimensional nuclear magnetic resonance (NMR) and capillary electrophoresis (CE). Characteristic fragmentation patterns for these compounds were obtained from ESI/MSn (with n = 2 to 4) experiments. IM-MS and CE analysis showed that a single component was mainly obtained for the complete grafting experimental conditions while a distribution of oligomers was produced for partially grafted products. The physical separation of GlynG1(N) oligomer ions was achieved in the gas phase (IM-MS) as well as in the condensed phase (CE). Besides, the collision cross sections (CCS) were estimated by IM-MS and compared to theoretical values. Then, the glycine grafting yield for GlynG1(N) PAMAM was determined by both NMR and IM-MS experiments

A Unique (3+2) Annulation Reaction between Meldrum's Acid and Nitrones: Mechanistic Insight by ESI-IMS-MS and DFT Studies

International audienceThe fragile intermediates of the domino process leading to an isoxazolidin‐5‐one, triggered by unique reactivity between Meldrum's acid and an N‐benzyl nitrone in the presence of a Brønsted base, were determined thanks to the softness and accuracy of electrospray ionization mass spectrometry coupled to ion mobility spectrometry (ESI‐IMS‐MS). The combined DFT study shed light on the overall organocatalytic sequence that starts with a stepwise (3+2) annulation reaction that is followed by a decarboxylative protonation sequence encompassing a stereoselective pathway issue

Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenn