Preparation of 3-O-, 5-O- and N-palmitoyl derivatives of fumonisin B1 toxin and their characterisation with NMR and LC-HRMS methods.

We have previously published six esterified O-acyl (EFB1) and three N-acyl fumonisin B1 derivatives extracted from rice cultures inoculated with Fusarium verticillioides, amongst these the identification of N-palmitoyl-FB1 has been clearly established in a spiking experiment. At that time, it was assumed that as in the case of O-acyl-FB1 derivatives, linoleic-, oleic- or palmitic acid esterify through the OH group on the 3C or 5C atom of the carbon chain of the fumonisins. In our most recent experiments, we have synthetically acylated the FB1 toxin and subsequently purified 3-O-palmitoyl- and 5-O-palmitoyl-FB1 toxins in addition to the N-palmitoyl-FB1 toxin. They were identified and characterised using 1H and 13C NMR as well as LC-HRMS. Our aim was the identification of the previously detected O-acyl-FB1 derivatives over the course of a spiking experiment, which were obtained through the solid-phase fermentation of Fusarium verticillioides. By spiking the three synthesized and identified components one-by-one into the fungal culture extract and analysing these cultures using LC-MS, it was clearly demonstrated that the F. verticillioides strain produced both the 5-O-palmitoyl-FB1 and N-palmitoyl-FB1 toxins, but did not produce 3-O-palmitoyl-FB1. Thus, it is highly probable that the components thought to be 3-O-acyl-(linoleoyl-, oleoyl-, palmitoyl-) FB1 derivatives in our previous communication are presumably 10-O-acyl-FB1 derivatives. Since these acylated FB1 derivatives can occur naturally in e.g. maize, the use of these synthesized components as reference materials is of great importance in order to obtain accurate qualitative and quantitative data on the occurrence of acylated fumonisins in different matrices including maize based feed samples. The production of these substances has also made it possible to test their toxicity in cell culture and small animal experiments.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Special lipid-based diets alleviate cognitive deficits in the APPswe/PS1dE9 transgenic mouse model of Alzheimer's disease independent of brain amyloid deposition.

Dietary fish oil, providing n3 polyunsaturated fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), associates with reduced dementia risk in epidemiological studies and reduced amyloid accumulation in Alzheimer mouse models. We now studied whether additional nutrients can improve the efficacy of fish oil in alleviating cognitive deficits and amyloid pathology in APPswe/PS1dE9 transgenic and wild-type mice. We compared four isocaloric (5% fat) diets. The fish oil diet differed from the control diet only by substituted fish oil. Besides fish oil, the plant sterol diet was supplemented with phytosterols, while the Fortasyn diet contained as supplements precursors and cofactors for membrane synthesis, viz. uridine-monophosphate; DHA and EPA; choline; folate; vitamins B6, B12, C and E; phospholipids and selenium. Mice began the special diets at 5 months and were sacrificed at 14 months after behavioral testing. Transgenic mice, fed with control chow, showed poor spatial learning, hyperactivity in exploring a novel cage and reduced preference to explore novel odors. All fish-oil-containing diets increased exploration of a novel odor over a familiar one. Only the Fortasyn diet alleviated the spatial learning deficit. None of the diets influenced hyperactivity in a new environment. Fish-oil-containing diets strongly inhibited ß- and γ-secretase activity, and the plant sterol diet additionally reduced amyloid-ß 1-42 levels. These data indicate that beneficial effects of fish oil on cognition in Alzheimer model mice can be enhanced by adding other specific nutrients, but this effect is not necessarily mediated via reduction of amyloid accumulation.

Cloning and sequencing of cDNA encoding for the testis-specific fox (Vulpes vulpes) sperm polypeptide Vb of the cytochrome C oxidase.

Identification of fox (Vulpes vulpes) sperm antigens was carried out to assess their interest as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP8, a fox sperm protein of 14.7 kd. fSP8 was isoantigenic in foxes, as it was recognized by sera of both male and female foxes immunized with fox sperm proteins. No glycosylation was detected, on fSP8, as shown both by deglycosylation assay and lectin labeling. To determine the fSP8 sequence, the NH2-terminal sequence was first obtained, and a piece of cDNA was amplified from testicular RNA by Rapid Amplification of cDNA extremities polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 879 base pairs was obtained, which includes a major open reading frame coding for 128 amino acids. Mass spectrometric analyses have confirmed the position of the open reading frame. Analysis of the predicted amino acids sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated a homology with the Zinc binding site of the subunit Vb of the cytochrome c oxidase. On the C-terminal extremity, fSP8 presents a high homology to the Vb polypeptide of the cytochrome c oxidase from bovine, mouse, and human; however the 34 amino acids on the NH2-extremity were specific to fSP8. Moreover, it was demonstrated that this sequence was testis-specific. This could contribute to the antigenicity of this protein. fSP8 is one of the first fox sperm antigens to be cloned and sequenced.

Identification of a new, testis-specific sperm antigen localized on the principal piece of the spermatozoa tail in the fox (Vulpes vulpes).

Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH2-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RIIalpha domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.