Sequence similarity-driven proteomics in organisms with unknown genomes by LC-MS/MS and automated de novo sequencing

LC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, combined with data processing, stringent, and sequence-similarity database searching tools, was employed in a layered manner to identify proteins in organisms with unsequenced genomes. Highly specific stringent searches (MASCOT) were applied as a first layer screen to identify either known (i.e. present in a database) proteins, or unknown proteins sharing identical peptides with related database sequences. Once the confidently matched spectra were removed, the remainder was filtered against a nonannotated library of background spectra that cleaned up the dataset from spectra of common protein and chemical contaminants. The rectified spectral dataset was further subjected to rapid batch de novo interpretation by PepNovo software, followed by the MS BLAST sequence-similarity search that used multiple redundant and partially accurate candidate peptide sequences. Importantly, a single dataset was acquired at the uncompromised sensitivity with no need of manual selection of MS/MS spectra for subsequent de novo interpretation. This approach enabled a completely automated identification of novel proteins that were, otherwise, missed by conventional database searches.     

Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone

In this paper, we present results of the first comprehensive study of the introgression of both autosomal and sex-chromosome markers across the central European portion of the hybrid zone between two house mouse subspecies, Mus musculus musculus and M. m. domesticus. More than 1800 individuals sampled from 105 sites were analyzed with a set of allozyme loci (hopefully representing neutral or nearly neutral markers) and X-linked loci (which are assumed to be under selection). The zone center is best modeled as a single straight line independent of fine-scale local geographic or climatic conditions, being maintained by a balance between dispersal and selection against hybrids. The width (w) of the multilocus autosomal cline was estimated as 9.6 km whereas the estimate for the compound X-chromosome cline was about 4.6 km only. As the former estimate is comparable to that of the Danish portion of the zone (assumed to be much younger than the central European one), zone width does not appear to be related to its age. The strength (B) of the central barrier was estimated as about 20 km; with dispersal (sigma) of about 1 km/gen(1/2), this means effective selection (s*) is approximately 0.06-0.09 for autosomal loci and about 0.25 for X-linked loci. The number of loci under selection was estimated as N= 56-99 for autosomes and about 380 for X-linked loci. Finally, we highlight some potential pitfalls in hybrid zone analyses and in comparisons of different transects. We suggest that conclusions about parts of the mouse genome involved in reproductive isolation and speciation should be drawn with caution and that analytical approaches always providing some estimates should not be used without due care regarding the support or confidence of such estimates, especially if conclusions are based on the difference between these estimates. Finally, we recommend that analysis in two-dimensional space, dense sampling, and rigorous treatment of data, including inspection of likelihood profiles, are essential for hybrid zone studies.     

The effects of membrane compartmentalization of csk on TCR signaling

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.     

Linoleic acid-induced ultra-weak photon emission from Chlamydomonas reinhardtii as a tool for monitoring of lipid peroxidation in the cell membranes

Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes.     

Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis

Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas?vaginalis. The kinetic properties and thermostability of T.?vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T.?vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.     

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)1

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Family of CNP neuropeptides: common morphology in various invertebrates

Neuropeptides expressed in the command neurons for withdrawal behavior were originally detected in the the central nervous system (CNS) of the terrestrial snail Helix (command neurons peptides, CNP). The family of CNP-like neuropeptides bears a C-terminal signature sequence Tyr-Pro-Arg-X. Using antisera against two of them, we have studied the CNS of various invertebrates belonging to the phyla of mollusks, annelids and insects. The immunoreactive neurons were detected in all studied species. Stained neurons were either interneurons projecting along the CNS ganglia chain, or sensory neurons, or neurohormonal cells. Beyond common morphological features, the immunoreactive cells had another similarity: the level of CNP expression depended on the functional state of the animal. Thus, the homologous neuropeptides in evolutionary distant invertebrate species possess some common morphological and functional features.     

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

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Investigating species boundaries using DNA and morphology in the mite <Emphasis Type="Italic">Tyrophagus curvipenis</Emphasis> (Acari: Acaridae), an emerging invasive pest,with a molecular phylogeny of the genus <Emphasis Type="Italic">Tyrophagus</Emphasis>

Mites of the genus Tyrophagus (Acari: Acaridae) are among the most widespread and common mites, inhabiting diverse natural and anthropogenic habitats. Some species are pests of agricultural products and stored food and/or live in house dust, causing allergies to humans. We sequenced 1.2 kb of the mitochondrial COI gene for 38 individuals belonging to seven species of Tyrophagus, including T. curvipenis, T. putrescentiae, T. fanetzhangorum, T. longior, T. perniciosus, and T. cf. similis. Molecular phylogenetic analyses (1) recovered two major clades corresponding to the presence or absence of eyespots, and (2) separated all included morphological species. Tyrophagus curvipenis and T. putrescentiae had the lowest between-species genetic distances (range, mean?±?SD): 14.20–16.30, 15.17?±?0.40 (K2P). The highest within-species variation was found in T. putrescentiae 0.00–4.33, 1.78?±?1.44 (K2P). In this species, we recovered two distinct groups; however, no geographical or ecological dissimilarities were observed between them. Based on our analyses, we document important morphological differences between T. curvipenis and T. putrescentiae. For the first time, we record the occurrence of T. curvipenis in the New World and suggest that it may be an emerging pest as it is currently spreading in agricultural produce.