Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

Seasonal and substrate preferences of fungi colonizing leaves in streams: traditional versus molecular evidence

Aquatic hyphomycetes are the main fungal decomposers of plant litter in streams. We compared the importance of substrate (three leaf species, wood) and season on fungal colonization. Substrates were exposed for 12 4-week periods. After recovery, mass loss, fungal biomass and release of conidia by aquatic hyphomycetes were measured. Fungal communities were characterized by counting and identifying released conidia and by extracting and amplifying fungal DNA (ITS2), which was subdivided into phylotypes by denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP). Mass loss, fungal biomass and reproduction were positively correlated with stream temperature. Conidial diversity was highest between May and September. Numbers of different phylotypes were more stable. Principal coordinate analyses (PCO) and canonical analyses of principal coordinates (CAP) of presence/absence data (DGGE bands, T-RFLP peaks and conidial species) showed a clear seasonal trend (Por=0.88). Season was also a significant factor when proportional similarities of conidial communities or relative intensities of DGGE bands were evaluated (P相似文献   

Determining diversity of freshwater fungi on decaying leaves: comparison of traditional and molecular approaches

Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of Articulospora tetracladia dominated. Modifications of two molecular methods-denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis-suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of A. tetracladia. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams.     

Antiproliferative activity of 2,3-disubstituted indoles toward apoptosis-resistant cancers cells

Many types of cancer, including glioma, melanoma, NSCLC, among others, are resistant to apoptosis induction and poorly responsive to current therapies with propaptotic agents. We describe a series of 2,3-disubstituted indoles, which display cytostatic rather than cytotoxic effects in cancer cells, and serve as a new chemical scaffold to develop anticancer agents capable of combating apoptosis-resistant cancers associated with dismal prognoses.     

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.     

Regulation of mitochondrial NADP-isocitrate dehydrogenase in rat heart during ischemia

The changes in the regulation of at mitochondrial NADP-isocitrate dehydrogenase (NADP-ICDH) in a rat heart during have been analysed. Increase of enzyme activity in the cytosol and mitochondria of the heart ischemia was detected. Catalytic properties of the mitochondrial NADP-ICDH at norm and pathology have been compared on homogeneous enzyme preparations. Enzyme from the normoxic and ischemic heart showed the same electrophoretical mobility and molecular mass. Enzyme isolated from the ischemic heart mitochondria demonstrated higher activation energy and lower thermal stability. NADP-isocitrate dehydrogenase at the normoxic and ischemic conditions exhibited different K_m for substrates and regulatory behaviour in relation to ATP, ADP, 2-oxoglutarate, citrate, malate, reduced and oxidised glutathione. The inhibitory effect of the Fe²⁺ and H₂O₂ mixture associated with the generation of hydroxyl radicals was lower in the ischemic enzyme. We hypothesise that the specific features of regulation behaviour of NADP-ICDH from the ischemic tissues permits the enzyme to supply NADPH to the glutathione reductase/glutathione peroxidase system.     

Pore formation by Vibrio cholerae cytolysin requires cholesterol in both monolayers of the target membrane

Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes, the rate of pore formation is limited by the leaflet with the lower cholesterol content. Methyl-beta-cyclodextrin, which removes cholesterol from membranes, rapidly inhibits VCC pore formation, even when it is added to the side opposite that of VCC addition. The results suggest that cholesterol in both membrane leaflets aid VCC-pore formation and that either leaflet can function as a kinetic bottleneck with respect to the rate of pore-formation.     

Association of G-quadruplex forming sequences with human mtDNA deletion breakpoints

Background

Mitochondrial DNA (mtDNA) deletions cause disease and accumulate during aging, yet our understanding of the molecular mechanisms underlying their formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are associated with nuclear DNA instability in cancer; recent evidence indicates they can also form in mitochondrial nucleic acids, suggesting that these non-B DNA structures could be associated with mtDNA deletions. Currently, the multiple types of GQ sequences and their association with human mtDNA stability are unknown.

Results

Here, we show an association between human mtDNA deletion breakpoint locations (sites where DNA ends rejoin after deletion of a section) and sequences with G-quadruplex forming potential (QFP), and establish the ability of selected sequences to form GQ in vitro. QFP contain four runs of either two or three consecutive guanines (2G and 3G, respectively), and we identified four types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position of each motif set relative to either 5'' or 3'' unique mtDNA deletion breakpoints, and found that intrastrand QFP sequences, but not ddi QFP sequences, showed significant association with mtDNA deletion breakpoint locations. Moreover, a large proportion of these QFP sequences occur at smaller distances to breakpoints relative to distribution-matched controls. The positive association of 2G QFP sequences persisted when breakpoints were divided into clinical subgroups. We tested in vitro GQ formation of representative mtDNA sequences containing these 2G QFP sequences and detected robust GQ structures by UV–VIS and CD spectroscopy. Notably, the most frequent deletion breakpoints, including those of the "common deletion", are bounded by 2G QFP sequence motifs.

Conclusions

The potential for GQ to influence mitochondrial genome stability supports a high-priority investigation of these structures and their regulation in normal and pathological mitochondrial biology. These findings emphasize the potential importance of helicases that subsequently resolve GQ to maintain the stability of the mitochondrial genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-677) contains supplementary material, which is available to authorized users.