Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.     

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ～90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.     

Ceramide profiles in the brain of rats with diabetes induced by streptozotocin

Diabetes is associated with disturbances of brain activity and cognitive impairment. We hypothesize that ceramides may constitute an important contribution to diabetes-linked neuro-dysfunction. In our study we used rats injected with streptozotocin (STZ) as a model of severe hyperglycemia. Using the gas-liquid chromatography technique we found a significant increase of ceramide content in brains and a decrease in plasma of diabetic rats. The inhibitor of serine palmitoyltransferase, myriocin, reduced ceramide generation in hyperglycemic brains, although injected alone it exerted a paradoxical effect of ceramide upregulation. Myriocin had no impact on ceramide concentration in the plasma of either control or diabetic rats. The level of ceramide saturated fatty acids was elevated whereas the level of ceramide poly-unsaturated fatty acids was downregulated in brains of all experimental groups. The concentration of ceramide mono-unsaturated fatty acids remained unchanged. The pattern of individual ceramide species was altered depending on treatment. We noted an STZ-evoked increase of brain ceramide C16:0, C18:0 and C20:0 and a strong decline in ceramide C18:2 fatty acid levels. Some changes of brain ceramide pattern were modified by myriocin. We found a decreased amount of total ceramide-ω-6 fatty acids in STZ-treated rat brains and no changes in ceramide-ω-3 concentration. We conclude that ceramides may be important mediators of diabetes-accompanied brain dysfunction.     

MHC class II derived recombinant T cell receptor ligands protect DBA/1LacJ mice from collagen-induced arthritis

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.     

Decrease in sex difference in premature mortality during system transformation in Poland

In recent years, sex differences in mortality have followed diverse patterns of change in developed countries. As there is no analogous evidence from Poland, the aim of this study was to describe the pattern of change in excess male mortality among Polish inhabitants aged 35-64 during 1995-2002, when the major socioeconomic transformation occurred, and compare it with sex differences in mortality observed in the late 1980s. During the study period, excess male mortality decreased significantly, independently of age and educational level. The reduction in mortality was observed in both sexes, but its magnitude was greater in men. These changes resulted mainly from a reduction in mortality rates due to cardiovascular disease and lung cancer in males and a concomitant increase in mortality rates due to lung cancer and suicides in females. Although, in general, excess male mortality decreased, social gradients related to this phenomenon increased. Subjects (in particularly men) who had graduated from university benefited the most, their magnitude of reduction in mortality rates being the greatest. Changes in social environment during the transformation period in Poland are suggested as major determinants of these changes, but further studies are needed.     

Slow vacuolar channels in vacuoles from winter and spring varieties of rape (Brassica napus)

Currents passing through slowly activating vacuolar channels (SV) in isolated vacuoles from winter (Górczański) and spring (M?ochowski) varieties of rape (Brassica napus) were examined using the patch-clamp technique. Eight-week-long vernalization at 5/2 degrees C (day/night) was applied to obtain the generative stage of winter rape. SV channels of vacuoles isolated from vegetative (rosette) and generative leaves of both varieties were examined in order to investigate a possible role of these ion channels in rape flowering. Single SV channel conductance measured in a vacuole-attached configuration (natural cell sap) ranged from 60 to 83 pS. Lower values were observed in the generative leaves of both varieties. Unitary conductance measured in excised cytoplasm-out membrane patches did not differ significantly among the experimental variants, with the exception of spring generative vacuoles, where it was significantly lower. There was also no difference in SV current densities measured in the whole-vacuole configuration. Gibberellic acid (GA(3)) (2mg/l) caused lowering of macroscopic SV currents by 20%, and had no significant effect on the single channel conductance. We conclude that SV channels play a role in rape vernalization and flowering owing to their multifactor regulation abilities rather than structural changes.     

Cellular and transcriptomic analysis of NS0 cell response during exposure to hypoxia

Mammalian cells have the ability to alter their gene expression in order to survive or adapt to a variety of environment stresses including hypoxic stress. Maintaining oxygen supply has been accepted as essential for cell survival and growth. To determine the cellular and molecular changes which take place under oxygen deprivation, an NS0 cell line producing a human-mouse chimeric antibody was cultured under hypoxic conditions (<1%). Various cellular parameters such as viability, productivity, metabolism, apoptosis and cell cycle were studied and notable changes were shown to be accompanied by changes in metabolic rates. When the cells where exposed to hypoxia for 48 h, cell growth was suppressed and cell death was detected. To better understand and explore the mechanisms underpinning these biological alterations and to identify the genes involved in the genetic reprogramming, genome-wide analyses were performed using GeneChip Mouse Genome arrays. The gene expression profiling generated by the microarray technique revealed that hypoxia, even in the early stages (12h), induces significant changes in gene expression in NS0 cells. The primary responses to hypoxia within the cells were: (1) the up-regulation of pathways such as glycolysis that ultimately lead to alternative routes of ATP generation and increased oxygen availability; and (2) the down-regulation of genes involved in purine/pyrimidine and one carbon pool metabolisms required for DNA and RNA synthesis. By combining gene expression and physiological changes under hypoxia, it was possible to explore the mechanisms of hypoxia-induced alterations in more depth.     

Biofilm production by clinical strains of Acinetobacter baumannii isolated from patients hospitalized in two tertiary care hospitals

Microbial biofilms are considered as virulence factors. During the present study, 34 clinical strains of Acinetobacter baumannii, isolated from patients hospitalized in two tertiary care hospitals, were examined for biofilm formation. These strains showed high variability in biofilm formation. Furthermore, no relation could be found between the ability of biofilm production and molecular type, carbapenem resistance, site of isolation of the clinical strains of A. baumannii and disease severity. Interestingly, in two cases an increase in biofilm formation could be detected in A. baumannii isolates cultured from the same patient upon prolonged hospitalization.     

Diterpenoid production in hairy root culture of Salvia sclarea L

Growth and diterpenoid accumulation (salvipisone, ferruginol, aethiopinone and 1-oxoaethiopinone) during the growth cycle of a Salvia sclarea hairy root culture are described. The roots transformed by Agrobacterium rhizogenes (LBA 9402) were cultured in half-strength B5 liquid medium supplemented with 30 g L(-1) sucrose under light (16 h/8 h light/dark). A culture period of 30 days was optimal for both biomass and diterpenoid production. The total content of four diterpenoids in the hairy roots [(27.3 +/- 0.6) mg g(-1) dry weight] was higher than that of roots of field-grown S. sclarea plants [(3.15 +/- 0.15) mg g(-1) dry weight]. In transformed roots, aethiopinone was the main diterpenoid, whereas the principal diterpenoid of natural roots was salvipisone.     

Correlation between chloroplast motility and elastic properties of tobacco mesophyll protoplasts

Translocations of chloroplasts induced by blue light were investigated in both leaves and protoplasts isolated from leaf mesophyll of Nicotiana tabacum. In the leaf tissue, the responses of chloroplasts were similar to those observed in other, higher and lower plant species. Weak and strong light induced movements of chloroplasts towards cell walls perpendicular and parallel to the light direction, respectively. Treatment with cytochalasin D, an actin-disturbing agent, blocked the movements. This shows that actin is involved in the motile system of chloroplast translocation in tobacco. By monitoring the response of chloroplasts to light in isolated protoplasts, we addressed the question whether the presence of the cell wall is necessary for the translocations of chloroplasts to occur. In control protoplasts (isolated at room temperature from unstressed leaves), no clear light intensity-dependent changes were observed in chloroplast distribution pattern. In contrast, in protoplasts obtained from plants treated with 4 °C for 8 h the chloroplasts maintained their responsiveness to light. Atomic Force Microscopy was used to measure elastic properties of the protoplasts. Young’s modulus, which reflects rigidity of the material, was 10 times higher for protoplasts of the coldstressed plants as compared to those isolated from the control plants. The rigidity of protoplasts isolated from the plants treated with low temperature was reduced four-fold by exposure to cytochalasin D. It appears that the status of protoplast actin is a factor responsible for elasticity of protoplasts. We speculate that unknown, cold stress-induced factors, maintain the orientational movements due to anchorage of the actin cytoskeleton in the plasma membrane despite the cell wall removal.