Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells

Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN(-/-) cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN(-/-) cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency.     

Glucocorticoid-receptor complexes in rat thymus cells. Rapid kinetic behavior and a cyclic model

We have studied the kinetics, on time scales of minutes and seconds, of formation and interconversion of glucocorticoid-receptor complexes in rat thymus cells under physiological conditions. Nonactivated and activated complexes were measured by a minicolumn technique that permits rapid, multiple simultaneous assays. The rate-limiting step in formation of nuclear complexes was activation, which at 37 degrees C had a half-time of 30-60 s. Activation in cells at 25 degrees C followed first order kinetics. Nuclear binding at 37 degrees C was too fast to measure, and probably has a half-time below 10 s. Earlier findings suggesting that triamcinolone acetonide and dexamethasone give higher steady state ratios of activated to nonactivated complexes than cortisol and corticosterone have been supported by showing that these ratios are concentration-independent, and are unlikely to be due to degradation or dissociation of complexes after cell disruption. A simple cyclic model of receptor kinetics, in which each glucocorticoid is characterized by its dissociation rate constant, accounts quantitatively for these results and many others. The model is based on the assumptions that activation is irreversible, and that energy is required for regenerating functional receptors after each cycle. It yields steady state ratios of activated to nonactivated complexes in agreement with experiment without introducing steroid-specific allosteric influences on activation, and suggests a new mechanism for explaining agonist-antagonist relationships.     

Emergence of white pine needle damage in the northeastern United States is associated with changes in pathogen pressure in response to climate change

The defoliation of the eastern white pine (Pinus strobus) across the northeastern United States is an escalating concern threatening the ecological health of northern forests and economic vitality of the region's lumber industry. First documented in the spring of 2010 affecting 24 328 hectares in the state of Maine, white pine needle damage (WPND) has continued to spread and is now well established in all New England states. While causal agents of WPND are known, current research is lacking in both sampling distribution and the specific environmental factor(s) that affect the development and spread of this disease complex. This study aims to construct a more detailed distribution map of the four primary causal agents within the region, as well as utilize long‐term WPND monitoring plots and data collected from land‐based weather stations to develop a climatic model to predict the severity of defoliation events in the proceeding year. Sampling results showed a greater distribution of WPND than previously reported. WPND was generally found in forest stands that compromised >50% eastern white pine by basal area. No single species, nor a specific combination of species had a dominating presence in particular states or regions, thus supporting the disease complex theory that WPND is neither caused by an individual species nor by a specific combination of species. In addition, regional weather data confirmed the trend of increasing temperature and precipitation observed in this region with the previous year's May, June, and July rainfall being the best predictor of defoliation events in the following year. Climatic models were developed to aid land managers in predicting disease severity and accordingly adjust their management decisions. Our results clearly demonstrate the role changing climate patterns have on the health of eastern white pine in the northeastern United States.     

Adenosine induces growth-cone turning of sensory neurons

The formation of appropriate connections between neurons and their specific targets is an essential step during development and repair of the nervous system. Growth cones are located at the leading edges of the growing neurites and respond to environmental cues in order to be guided to their final targets. Directional information can be coded by concentration gradients of substrate-bound or diffusible-guidance molecules. Here we show that concentration gradients of adenosine stimulate growth cones of sensory neurons (dorsal root ganglia) from chicken embryos to turn towards the adenosine source. This response is mediated by adenosine receptors. The subsequent signal transduction process involves cAMP. It may be speculated that the in vivo function of this response is concerned with the formation or the repair and regeneration of the peripheral nervous system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

A ratiometric imaging method for mapping ion flux densities

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiometric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the local influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratioing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux density distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution patterns of ion channels active during a given stimulus may be derived.     

Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody

The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.     

Preparation and properties of a new type of acyclic, achiral nucleoside analogue

Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by Tm measurements showing a deltaTm of -2 to -6.5 degrees C per modification. An oligonucleotide with two modifications at the 3'-end showed considerable resistance towards cleavage by a 3'-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.     

Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.