Non-histone protein methylation in Trypanosoma cruzi epimastigotes

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.     

Proteomics in Drosophila melanogaster.

To be able to understand cellular mechanisms, we require fully integrated data sets combining information about gene expression, protein expression, post-translational modification states, sub-cellular location and complex formation. Proteomics is a very powerful technique that can be applied to interrogate changes at the protein level. Studying this effectively requires specialised facilities within research institutes. Here, we describe the setting up and operation of such a facility, providing a resource for the Arabidopsis and Drosophila research communities.     

FAB CID-MS/MS characterization of tetrasaccharide tri- and tetrasulfate derived from the antigenic determinant recognized by the anti-chondroitin sulfate monoclonal antibody MO-225

The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MS/MS mass spectrometry/mass spectrometry - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose     

Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction.

Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.     

互叶白千层油化学成分的研究

采用水蒸汽蒸馏收集互叶白千层芳香油,对该油进行ＧＣ／ＭＳ／ＤＳ定性分析及其总离于流图的面积归一化定量分析,鉴定出２５种化合物,其中含氧化合物６种,碳氢化合物１９种。直接影响该油商品价值的化学成分４－萜品醇含量约为１７％,桧樟脑含量约为２．４％。     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography-mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.     

Mass spectrometry of silylated flavonol O-glycosides

The electron impact mass spectra of 19 trimethyl silylated flavonol mono-, di- and -triglycosides are reported for the first time. All spectra show wel     

Bacteriophage MS2 RNA: A correlation between the stability of the codon: Anticodon interaction and the choice of code words

Summary The non-random distribution of degenerate code words in Bacteriophage MS2 RNA can be explained partially by considerations of the stability of the codon-anticodon complex in prokaryotic systems. Supporting this hypothesis we note that wobble codons are positively selected in codons having G and/or C in the first two positions. In contrast, wobble codons are statitically less likely in codons composed of A and U in the first two positions. Analyses of nucleotides adjacent to 5 and 3 ends of codons indicate a nonrandom distribution as well. It is thus likely that some elements of RNA evolution are independent of the structural needs of the RNA itself and of the translated protein product.This work was supported by grants from the Belgian Government Actions concertées - Gekon-certéerde Acties, N.F.W.O. and F.K.F.O. as well as from le Ministère de l'éducation du Québec. A preliminary report of this work was given at the EMBO ribosome workshop, Brussels 1976     

Chromatographic resolution of amino acid adducts of aliphatic halides

Numerous xenobiotics are known to be bioactivated and to covalently bind to proteins, but the resulting amino acid adducts (AAAs) are unknown. In this study the AAAs of twelve ¹⁴C-labeled aliphatic halides were examined after formation in an in vitro microsomal system. After exhaustive solvent extraction of the precipitated microsomal protein, the AAAs were isolated by Pronase digestion, followed by filtration through a 500 mol. wt. exclusion membrane. The liberated AAAs were applied to a constant flow DC-4A cation exchange column, resolved by stepwise buffer elution, collected and counted for radioactivity. Column recovery for applied radioactivity was 100 ± 4%. Generally, 1–4 different AAAs (defined by eluting radioactivity) were resolved, with each organohalogen displaying a characteristic elution profile. Methyl iodide, trichloroethylene and 1,2-dichloroethylene had a single major AAA while bromotrichloromethane, 1,2-dibromoethane, 1,1,1-trichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, chloroform and carbon tetrachloride had up to 4 AAAs or more, indicating combinations of binding site(s) and reactive intermediate(s). The single AAA formed following incubation of methyl iodide with the microsomes was identified as S-methylcysteine. Thus, this method appears capable of resolving binding sites and is the initial isolation step for identifying specific adducts to proteins.