Molecular Insights into the Architecture of the Human SMC5/6 Complex.

A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular processes ensuring proper cohesion, condensation and replication. They share a common SMC-kleisin architecture allowing them to embrace DNA. In SMC5/6, the NSE1 and NSE3 KITE and NSE4 kleisin subunits form a stable subcomplex that binds DNA and regulates essential processes. In addition, NSE5 and NSE6 subunits associate with the core SMC5/6 complex and recruit it to DNA repair sites. The architecture of the SMC5/6 complex is crucial for its proper functioning, and mutations within the human SMC5/6 subunits result in severe syndromes. Therefore, we aimed to analyze interactions within the human SMC5/6 complex and determine its detailed architecture. Firstly, we analyzed different parts of SMC5/6 by crosslinking and MS/MS analysis. Our data suggested domain arrangements of hNSE1-hNSE3 and orientation of hNSE4 within the hNSE1-hNSE3-hNSE4 subcomplex. The crosslinking and electron microscopic analysis of the SMC5/6 core complex showed its rod-like architecture with juxtaposed hSMC5-hSMC6 arms. Additionally, we observed fully or partially opened hSMC5-hSMC6 shapes with the hNSE1-hNSE3-hNSE4 trimer localized in the SMC head domains. To complete mapping of the human SMC5/6 complex architecture, we analyzed positions of hNSE5-hNSE6 at the hSMC5-hSMC6 arms. We showed that hNSE6 binding to hNSE5 and the coiled-coil arm of hSMC6 is mediated by a conserved FAM178 domain, which we therefore renamed CANIN (Coiled-coil SMC6 And NSE5 INteracting) domain. Interestingly, hNSE6 bound both hSMC5 and hSMC6 arms, suggesting that hNSE6 may lock the arms and regulate the dynamics of the human SMC5/6 complex.

Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene.

Staphylococcus sciuri is a bacterial pathogen associated with infections in animals and humans, and represents a reservoir for the mecA gene encoding methicillin-resistance in staphylococci. No S. sciuri siphophages were known. Here the identification and characterization of two temperate S. sciuri phages from the Siphoviridae family designated ϕ575 and ϕ879 are presented. The phages have icosahedral heads and flexible noncontractile tails that end with a tail spike. The genomes of the phages are 42,160 and 41,448 bp long and encode 58 and 55 ORFs, respectively, arranged in functional modules. Their head-tail morphogenesis modules are similar to those of Staphylococcus aureus ϕ13-like serogroup F phages, suggesting their common evolutionary origin. The genome of phage ϕ575 harbours genes for staphylokinase and phospholipase that might enhance the virulence of the bacterial hosts. In addition both of the phages package a homologue of the mecA gene, which is a requirement for its lateral transfer. Phage ϕ879 transduces tetracycline and aminoglycoside pSTS7-like resistance plasmids from its host to other S. sciuri strains and to S. aureus. Furthermore, both of the phages efficiently adsorb to numerous staphylococcal species, indicating that they may contribute to interspecies horizontal gene transfer.

Proteome and transcript analysis of Vitis vinifera cell cultures subjected to Botrytis cinerea infection.

Gray mold caused by Botrytis cinerea is one of the most important diseases of grapevine resulting in significant reductions in yield and fruit quality. In order to examine the molecular mechanisms that characterize the interaction between B. cinerea and the host plant, the grapevine cytoplasmic proteome was analyzed by two-dimensional polyacrylamide gel electrophoresis. The interaction between Vitis vinifera cv. Gamay cells and B. cinerea was characterized by the increase in spot abundance of 30 proteins, of which 21 were successfully identified. The majority of these proteins were related to defence and stress responses and to cell wall modifications. Some of the modulated proteins have been previously found to be affected by other pathogens when they infect V. vinifera but interestingly, the proteins related to cell wall modification that were influenced by B. cinerea have not been shown to be modulated by any other pathogen studied to date. Transcript analysis using the quantitative real time polymerase chain reaction additionally revealed the up-regulation of several acidic, probably extracellular, chitinases. The results indicate that cell wall strengthening, accumulation of PR proteins and excretion of lytic enzymes are likely to be important mechanisms in the defence of grapevine against B. cinerea. BIOLOGICAL SIGNIFICANCE: Although gray mold caused by Botrytis cinerea is one of the most important diseases of grapevine, little information is available about proteomic changes in this pathosystem. These results suggest that cell wall strengthening, accumulation of PR proteins and excretion of lytic enzymes are important molecular mechanisms in the defence of grapevine against B. cinerea. Surprisingly, the proteins related to cell wall modification that were modulated by B. cinerea have not been shown to be affected by any other pathogen studied to date.

Prey-race drives differentiation of biotypes in ant-eating spiders.

1. Disruptive natural selection resulting from specialization on different hosts is recognized as one of the most important driving forces in the diversification of herbivores and parasites. It has been proposed that a similar mechanism could apply to carnivorous predators too, although the evidence is still lacking. 2. Here, we show that the differentiation of biotypes of specialized ant-eating spiders of the genus Zodarion has probably been induced by prey-shifting. We focused on two forms of one species Z. styliferum from the Iberian Peninsula that presumably represent ecological races. We conducted geographic, ecological, venom-oriented, reproductive and genetic divergence analysis among multiple populations collected at a number of sites across Portugal and Madeira. 3. Geographic analysis revealed that the two forms occur in mosaic sympatry. Each form was found to associate in nature with a different ant species in a different habitat. Specifically, the styliferum form hunted predominantly Messor ants, and the extraneum form hunted mainly Camponotus ants. Laboratory experiments revealed that the two forms exhibit a significant preference for attacking focal ants, demonstrating higher paralysis efficiency, and also show different venom composition. Cross-mating of the two forms was significantly less likely than between pairs of the same form, suggesting moderate assortative mating. Phylogenetic analyses indicate low genetic differentiation of the two forms and parallel-repeated evolution of biotypes. 4. Adaptive prey-shifting correlated with habitat preference are at present the most valid explanations for biotype formation in Zodarion. The speciation of ant-eating Zodarion spiders thus appears to follow a scenario similar to that of host-shifting in parasites and herbivores.

[Comparative plasma proteomic analysis of patients with multiple myeloma treated with bortezomib-based regimens]. / Srovnávací proteomická analýza krevní plazmy pacientu s mnohocetným myelomem lécených rezimy s bortezomibem.

BACKGROUNDS: Recently, the term biomarker has become, especially in connection with the term clinical proteomics, one of the most frequent terms in the field of biomedical research. The aim of this work was to select an appropriate pre-fractionation method of blood plasma prior to a subsequent proteomic analysis of low-abundant fraction of proteins by two dimensional gel electrophoresis (2-DE) and mass spectrometry to improve the resolution of 2-DE maps and protein identification. MATERIALS AND METHODS: First, we compared two prefractionation methods (MARS versus ProteoMiner) preceding 2-DE analysis using 10 blood plasma samples. Based on the results of the comparative experiments, low-abundant plasma protein fractions from 18 multiple myeloma patients treated with bortezomib were analyzed. Patients were divided into two groups: a group resistant to chemotherapy (9 patients--disease progression, stable disease) and a group with positive clinical response (9 patients--complete and partial remission). RESULTS AND CONCLUSION: Samples prefractioned by ProteoMiner method yielded 2-DE maps with a significantly increased number of detected protein spots, as compared to immunodepletion method MARS (Multiple Affinity Removal System). Between groups of chemoresistant and sensitive patients treated with bortezomib, 15 differently intense spots were revealed by image analysis. These spots were found to correspond to 10 proteins, as confirmed by mass spectrometry. Seven proteins had significantly lower protein level in the group of chemosensitive patients (serum amyloid P, fibrinogen--gamma chain, retinol-binding protein 4, complement factor C4-A, apolipoprotein E, carboxypeptidase N and complement factor H-related protein 1) and 3 proteins showed significantly higher levels of protein (or were only detected) in the group of chemosensitive patients (serum paraoxonase 1, alpha-1-antitrypsin and complement factor B).

A combined immunoprecipitation and mass spectrometric approach to determine deltaNp63-interacting partners.

Expression of p63 is essential for the formation of epidermis and other stratifying epithelia. Moreover p63 is highly expressed in several epithelial cancers and is involved in tumourigenesis and controlling chemo-sensitivity. The identification of p63 interacting partners is essential for understanding the complex network of gene regulation managing epithelial development and could also help to reveal signalling pathways participating in UV-damage response in human skin. We used a proteomic approach to identify proteins that interact with deltaNp63. Proteins were isolated by immunoprecipitation with deltaNp63 specific antibody and analysed by mass spectrometry. We identified 23 proteins as potential deltaNp63 binding partners that were not present in negative control samples. These results will be evaluated using other methods.

Fe/Mn superoxide dismutase-encoding gene in Paracoccus denitrificans is induced by azide and expressed independently of the FNR-type regulators.

Paracoccus denitrificans cells undergo changes in protein composition upon exposure to azide, a known activator of the fumarate-nitrate reduction (FNR)-type transcription factor NarR. One of the most prominent protein species inducible by azide is a Fe/Mn-family superoxide dismutase (SOD). Azide induces SOD at protein, mRNA transcript, and enzyme activity levels in the aerobically growing cells. Since SOD expression remains unaffected in the fnrP-, nnr-, and narR-mutant strains, we postulate a mechanism independent of the known FNR-type regulators but involving a redox signal arising from the respiratory chain.

[Proteomic analysis of cancer cells]. / Proteomická analýza nádorových bunek.

The rapid development of analytical instrumentation and methodical approaches in the course of the last two decades has significantly extended the possibilities of studying proteins in living systems. Proteomic analysis provides ever deeper insights into the molecular nature of biological processes in terms of qualitative and quantitative changes in protein composition in connection with the physiological and pathological states of the organism. Thus, proteomic analysis contributes to a better understanding of these processes and becomes a tool for the development and validation of diagnostic and therapeutic approaches. Thanks to recent achievements, the attention of cancer specialists is more and more focused on human proteome research. In this brief review we explain the principles of widely used proteomic techniques (gel electrophoresis, liquid chromatography, mass spectrometry analysis, protein array technologies) and show examples of their application in oncology, namely hematooncological diseases.

Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin.

BACKGROUND AIMS: Microvesicles (MV) shed from the plasma membrane of eukaryotic cells, including human embryonic stem cells (hESC), contain proteins, lipids and RNA and serve as mediators of cell-to-cell communication. However, they may also contain immunogenic membrane domains and infectious particles acquired from xenogenic components of the culture milieu. Therefore, MV represent a potential risk for clinical application of cell therapy. METHODS: We tested the ability of hESC and their most commonly used feeder cells, mouse embryonic fibroblasts (MEF), to produce MV. We found that hESC are potent producers of MV, whereas mitotically inactivated MEF do not produce any detectable MV. We therefore employed a combined proteomic approach to identify the molecules that constitute the major components of MV from hESC maintained in a standard culture setting with xenogenic feeder cells. RESULTS: In purified MV fractions, we identified a total of 22 proteins, including five unique protein species that are known to be highly expressed in invasive cancers and participate in cellular activation, metastasis and inhibition of apoptosis. Moreover, we found that hESC-derived MV contained the immunogenic agents apolipoprotein and transferrin, a source of Neu5Gc, as well as mouse retroviral Gag protein. CONCLUSIONS: These findings indicate that MV represent a mechanism by which hESC communicate; however, they also serve as potential carriers of immunogenic and pathogenic compounds acquired from environment. Our results highlight a potential danger regarding the use of hESC that have previously been exposed to animal proteins and cells.

Characterization of novel di- and tricarboxylic acids in fine tropical aerosols.

Three unknown di- and tricarboxylic acids were characterized in the fine size fraction of aerosols which were collected during the wet season in the Amazon basin (Rondonia, Brazil). For the structural characterization of the methyl esters of these unknown compounds, mass spectrometry with electron ionization (EI) and tandem mass spectral techniques combined with gas chromatographic (GC) separation were employed. Fragment and parent ion spectra were recorded during elution of the GC peaks by linked scanning of the B and E sectors in combination with high-energy collision-induced dissociation. The fragmentation patterns of significant ions in the first-order EI spectra were also obtained for nonanedioic acid, which was examined as a model compound. The compounds were tentatively identified as 4-acetyloxyheptanedioic acid and cis and trans isomers of 5-hexene-1,1,6-tricarboxylic acid. Since there were indications of biomass burning during the aerosol sampling the di- and tricarboxylic acids characterized in the present work could be markers for biomass burning. Furthermore, the characterization of di- and tricarboxylic acids in the fine size fraction of atmospheric aerosols may be important for assessing the effects of organic aerosols in cloud formation.

Pressurised liquid extraction of ketones of polycyclic aromatic hydrocarbons from soil.

Pressurised liquid extraction (PLE) was used in the extraction of three ketones of polycyclic aromatic hydrocarbons from the sample of a soil highly contaminated with polycyclic polyaromatic compounds. The choice of solvent was the only factor that considerably influenced the extraction efficiency of PLE under the conditions recommended in Method 3545A promulgated by the United States Environmental Protection Agency. The dichloromethane-ethanol solvent mixture was found to be the most efficient solvent. PLE using this mixture provided better recoveries of all analysed ketones relative to Soxhlet extraction.

Determination of 4-nitrocatechol in biodegradation samples by gas chromatography-mass spectrometry.

4-Nitrocatechol was identified as a product of transformation of 4-nitrophenol by bacterial strain Corynebacterium sp.8/3 using direct acetylation of biodegradation samples by acetic anhydride followed by GC-MS analysis. The identity of 4-nitrocatechol, in the form of diacetate, was confirmed by electron-impact spectra and spectra recorded under chemical ionization conditions (positive and negative modes). Negative-ion chemical ionization was used for quantification of 4-nitrocatechol in biodegradation samples in a concentration range of 1-25 mg/l.

[Use of a computer program in the diagnosis of brain tumors]. / Pouzití pocítacového programu v diagnostice mozkových nádoru.

The FEL-EXPERT was used to improve the diagnosis of brain tumors. Basic structure and decision process of the system were characterized. In a group of 70 testing cases the correct diagnosis was established at the 1st possibility in 83%, as the 1st or 2nd possibility in 91% and as the 1st or 2nd or 3rd possibility in 17%.