Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.     

Recent developments in the study of orchid mycorrhiza

Orchids are mycoheterotrophic during their seedling stage and in many species the dependency on fungi as a carbohydrate source is prolonged into adulthood. The mycobionts in orchid mycorrhiza belong in at least 5 major taxonomic groups of basidiomycetes. Traditional records have mainly focused on saprotrophic mycobionts but the participation of both ectomycorrhizal and parasitic fungi in orchid mycorrhiza has been corroborated. There is an increasing evidence of specific relationships between orchids and fungi, though usually not on a species-to-species level. Physiological compatibility demonstrated under artificial conditions, as in vitro, may be much broader, however. Recent development of field sowing techniques has improved the possibilities of evaluating orchid-fungal relations in an ecological context. Although the general nutrient flow in orchid mycorrhiza is well known, some questions remain regarding breakdown processes of fungi within orchid tissues, especially the ptyophagic syndrome that has recently been illustrated at the ultrastructural level for the first time.     

Etlingera kenyalang (Zhgiberaceae) — a new species from Sarawak, Borneo, with notes on its ecology and Iban ethnobotany

Etlingera kenyalang is described from southern Sarawak. Ecological notes are given including a possible pollinator and seed dispersers. The Iban ethnobotany of the species is discussed; the shoots and fruits are edible and the leaves are used ritually during the Gawai Kenyalang (Hornbill Festival).     

Infraspecific variation in Cerastium alpinum s. lat. (Caryophyllaceae) in Central Norway

Within Cerastium alpinum s. lat., several taxa have been circumscribed. Three infraspecific taxa are currently recognized in Nordic literature, but the opinions have varied about their taxonomic levels and relations to each other. Populations of these three taxa — alpinum, glabratum and lanatum — were investigated in Central Norway to elucidate taxonomic relationships on a regional scale. Morphometric analyses (PCA ordination) based on two data sets; one with 61 characters, and one without the 35 indumentum characters, gave different results. The former partly support the use of a higher rank for glabratum , but this analysis may put too much weight on indumentum characters. The reduced data set gave a more continuous transition between the three taxa, with no special status for glabratum. Crossing experiments between the taxa indicated full cross compatibility as far as relative seed set was concerned. Finally, vegetation analysis (DCA ordination) indicated that the three taxa have somewhat different ecological demands. This suggests that they, in spite of hybridization abilities, normally are maintained as separate entities due to habitat differences. A delimitation of three infraspecific taxa at the same level within C. alpinum s. lat. is thus supported by this study. Whether these taxa should be treated as subspecies or varieties strongly depends upon the definitions of these categories. Viewed in a wide geographical context, however, the arguments for subspecific rank are strengthened.     

The use of genetic markers to measure genomic response to selection in livestock

A method to measure genomic response to natural and artificial selection by means of genetic markers in livestock is proposed. Genomic response through several levels of selection was measured using sequential testing for distorted segregation of alleles among selected and nonselected sons, single-sperm typing, and a test with records for growth performance. Statistical power at a significance level of 0.05 was >0.5 for a marker linked to a QTL with recombination fractions 0, 0.10, and 0.20 for detecting genomic responses for gene effects of 0.6, 0.7, and 1.0 phenotypic standard deviations, respectively. Genomic response to artificial selection in six commercial bull sire families comprising 285 half-sib sons selected for growth performance was measured using 282 genetic markers evenly distributed over the cattle genome. A genome-wide test using selected sons was significant (P < 0.001), indicating that selection induces changes in the genetic makeup of commercial cattle populations. Markers located in chromosomes 6, 10, and 16 identified regions in those chromosomes that are changing due to artificial selection as revealed by the association of records of performance with alleles at specific markers. Either natural selection or genetic drift may cause the observed genomic response for markers in chromosomes 1, 7, and 17.     

Trafficking of the IKs‐Complex in MDCK Cells: Site of Subunit Assembly and Determinants of Polarized Localization

The voltage‐gated potassium channel K_V7.1 is regulated by non‐pore forming regulatory KCNE β‐subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current I_Ks. However, where the subunits assemble and which of the subunits determines localization of the I_Ks‐complex has not been unequivocally resolved yet. We employed trafficking‐deficient K_V7.1 and KCNE1 mutants to investigate I_Ks trafficking using the polarized Madin‐Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post‐endoplasmic reticulum compartment. We demonstrate that K_V7.1 targets the I_Ks‐complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical K_V7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that K_V7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of K_V7.1/KCNE complexes .     

Phospholipase A2 Sensitive Liposomes for Delivery of Small Interfering RNA (siRNA)

Small interfering RNA (siRNA) is potent and highly specific for gene silencing and there is currently a lot of enthusiasm for developing siRNA into a drug. However, for most therapeutic applications of siRNA, delivery systems are needed. These delivery systems have multiple requirements and should on one hand ideally be stable carriers protecting the siRNA from degradation and on the other hand assist the siRNA in overcoming membrane barriers for intracellular delivery to the cytosol. Long-circulating liposomes, which are sensitive to secretory phospholipase A₂ (sPLA₂) are feasible delivery systems for systemic administration of drugs due to their passive targeting to pathological tissue via the enhanced permeability and retention (EPR) effect and their site-specific, enzyme-triggered release of encapsulated drug in response to sPLA₂ which exists locally at elevated levels at, e.g,. sites of inflammation. However, recent data suggest that endosomal membrane destabilizing approaches could be addressed to design sPLA₂-sensitive liposomes as successful delivery systems for siRNA to the RNA interference pathway in the cytoplasm upon systemic administration.     

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2.     

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin‐based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin‐associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin‐based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.     

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter-GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.