Neuronal ceroid lipofuscinoses are connected at molecular level: interaction of CLN5 protein with CLN2 and CLN3

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative storage diseases characterized by mental retardation, visual failure, and brain atrophy as well as accumulation of storage material in multiple cell types. The diseases are caused by mutations in the ubiquitously expressed genes, of which six are known. Herein, we report that three NCL disease forms with similar tissue pathology are connected at the molecular level: CLN5 polypeptides directly interact with the CLN2 and CLN3 proteins based on coimmunoprecipitation and in vitro binding assays. Furthermore, disease mutations in CLN5 abolished interaction with CLN2, while not affecting association with CLN3. The molecular characterization of CLN5 revealed that it was synthesized as four precursor forms, due to usage of alternative initiator methionines in translation. All forms were targeted to lysosomes and the longest form, translated from the first potential methionine, was associated with membranes. Interactions between CLN polypeptides were shown to occur with this longest, membrane-bound form of CLN5. Both intracellular targeting and posttranslational glycosylation of the polypeptides carrying human disease mutations were similar to wild-type CLN5.     

Antigen-independent selection of intracellular stable antibody frameworks

The intracellular expression of highly specific antibody fragments ("intrabodies") in eukaryotes has a great potential in functional genomics and therapeutics. However, since the intracellular reducing environment prevents formation of the conserved intrachain disulfide bonds, most antibodies do not fold properly and are therefore inactive inside cells. The few antibodies that have been found to function in an intracellular environment and that have been characterized for their biophysical properties have generally shown a high degree of stability and solubility. Thus, for intracellular expression and application, very stable antibody frameworks are needed that can correctly fold even in the absence of disulfide bonds and that do not aggregate. Here, we present and discuss a novel method, named "Quality Control," which allows selection of stable and soluble antibody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibody fragments (scFvs) fused to a selectable marker that can control gene expression and cell growth. The activity of such a selectable marker fused to various scFvs that have been biophysically characterized correlated with the solubility and stability of the scFv moieties. This antigen-independent intrabody selection system was applied to screen scFv libraries for identifying stable and soluble frameworks, which subsequently served as acceptor backbones to construct intrabody libraries by randomization of hypervariable loops.     

Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.     

Changes of mitochondrial respiration,mitochondrial content and cell size after induction of apoptosis in leukemia cells

Mitochondrial damage with release of cytochrome c is implicated in cell death signalling pathways. To examine mitochondrial function in apoptotic cells, we applied high-resolution respirometry to human leukemia cells arrested in the G1- and S-phase by exposure to the glucocorticoid dexamethasone and nucleotide analogue gemcitabine. At 30% apoptosis, opposite effects were observed on respiratory capacity (71% and 131% of controls, respectively). These changes correlated with alterations in cell size, cytosolic, and mitochondrial marker enzymes. Mitochondrial ATP production and membrane potential were maintained in all treatments, as deduced from high respiratory uncoupling control ratios (UCR). Bcl-2 over-expression did not prevent apoptosis after gemcitabine-treatment, but protected dexamethasone-treated cells from apoptosis, without fully preventing the decline of respiration and cell size. These results, therefore, provide conclusive evidence that alterations in respiratory capacity and enzyme activities per cell are mainly caused by opposite changes in cell size, occurring upon cell cycle arrest triggered by dexamethasone and gemcitabine in the early phase of apoptosis.     

North Atlantic Oscillation and timing of spring migration in birds

Migrant birds have been trapped on the island of Helgoland (southeastern North Sea) since 1909, with methods and sampling effort remaining unchanged throughout the last four decades. In 12 short/medium-distance migrants and 12 long-distance migrants (23 passerines plus the European woodcock) sample sizes were sufficient to calculate mean spring passage (msp) times and to relate these to climate change. All but one species, passing Helgoland en route to their breeding areas (mainly in Scandinavia), show a trend towards earlier msp-time, which is significant in 7 short/medium-distance migrants and 10 long-distance migrants. The msp-times advanced by 0.05-0.28 days per year, short/medium-distance migrants not differing from long-distance migrants. In 23 out of the 24 species, earlier msp-times coincide with local warmer msp-temperatures (significantly in 11 and 7 species of the two groups, respectively). Even more striking is the relation to a large-scale phenomenon, the North Atlantic Oscillation (NAO), during the last four decades. Again, in 23 out of the 24 species, an earlier msp-time coincides with higher NAO indices (significantly in 9 and 12 species, respectively). The NAO index can also explain differences and similarities in spring migration strategies, as well as migration routes within Europe.     

Immunodominance of an antiviral cytotoxic T cell response is shaped by the kinetics of viral protein expression

Lymphocytic choriomeningitis virus (LCMV) infection induces a protective CTL response consisting of gp- and nucleoprotein (NP)-specific CTL. We find that a small load of LCMV led to immunodominance of NP-CTL, whereas a large viral load resulted in dominance of gp-CTL. This is the first study describing that immunodominance is not fixed after infection with a given pathogen, but varies with the viral load instead. We assumed higher Ag sensitivity for NP-CTL, which would explain their preferential priming at low viral load, as well as their overstimulation resulting in selective exhaustion at high viral load. The higher Ag sensitivity of NP-CTL was due to faster kinetics of NP-epitope presentation. Thus, we uncover a novel factor that impinges upon immunodominance and is related to the kinetics of virus protein expression. We propose that CTL against early viral proteins swiftly interfere with virus replication, resulting in efficient protection. If these "early" CTL fail in immediate virus control, they are activated in the face of higher viral load compared with "late" CTL and are therefore prone to be exhausted. Thus, the observed absence of early CTL in persistent infections might not be the cause, but rather the consequence of viral persistence.     

Radio frequency electromagnetic field exposure in humans: Estimation of SAR distribution in the brain,effects on sleep and heart rate

In two previous studies we demonstrated that radiofrequency electromagnetic fields (RF EMF) similar to those emitted by digital radiotelephone handsets affect brain physiology of healthy young subjects exposed to RF EMF (900 MHz; spatial peak specific absorption rate [SAR] 1 W/kg) either during sleep or during the waking period preceding sleep. In the first experiment, subjects were exposed intermittently during an 8 h nighttime sleep episode and in the second experiment, unilaterally for 30 min prior to a 3 h daytime sleep episode. Here we report an extended analysis of the two studies as well as the detailed dosimetry of the brain areas, including the assessment of the exposure variability and uncertainties. The latter enabled a more in depth analysis and discussion of the findings. Compared to the control condition with sham exposure, spectral power of the non-rapid eye movement sleep electroencephalogram (EEG) was initially increased in the 9-14 Hz range in both experiments. No topographical differences with respect to the effect of RF EMF exposure were observed in the two experiments. Even unilateral exposure during waking induced a similar effect in both hemispheres. Exposure during sleep reduced waking after sleep onset and affected heart rate variability. Exposure prior to sleep reduced heart rate during waking and stage 1 sleep. The lack of asymmetries in the effects on sleep EEG, independent of bi- or unilateral exposure of the cortex, may indicate involvement of subcortical bilateral projections to the cortex in the generation of brain function changes, especially since the exposure of the thalamus was similar in both experiments (approx. 0.1 W/kg).     

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.     

Identification of proteins involved in osmotic stress response in Enterobacter sakazakii by proteomics

Enterobacter sakazakii is considered an opportunistic food-borne pathogen, causing rare but significant illness especially in neonates. It has been proposed that the organism is relatively resistant to osmotic and dry stress compared to other species of the Enterobacteriaceae group. To understand the mechanisms involved in osmotic stress response, 2-DE protein analysis coupled to MALDI-TOF MS was employed to investigate changes in the protein profiles of E. sakazakii cells in response to two different types of osmotic stress (physical desiccation and growth in hyperosmotic media). In total, 80 differentially expressed protein spots corresponding to 53 different protein species were identified. Affiliation of proteins to functional categories revealed that a considerable number of the differentially expressed proteins from desiccated and hyperosmotic grown samples belonged to the same functional category but were regulated in opposite directions. Our data show that the protein pattern of NaCl-grown cultures reflect more or less a general down-regulation of central metabolic pathways, whereas adaptation of (non-growing) cells in a desiccated state represents an accumulation of proteins that serve some structural or protective role. The most striking effects observed for both types of osmotic stress in E. sakazakii were a significant down-regulation of the motility apparatus and the formation of filamentous cells.