Dynamic culture in a rotating-wall vessel bioreactor differentially inhibits murine T-lymphocyte activation by mitogenic stimuli upon return to static conditions in a time-dependent manner.

Depressed immune function is a well-documented effect of spaceflight. Both in-flight studies and ground-based studies using microgravity analogs, such as rotating wall vessel (RWV) bioreactors, have demonstrated that mitogen-stimulated T lymphocytes exhibit decreased proliferation, IL-2 secretion, and activation marker expression in true microgravity and the dynamic RWV-culture environment. This study investigates the kinetics of RWV-induced T lymphocyte inhibition by monitoring the ability of Balb/c mouse splenocytes to become activated under static culture conditions after concanavalin A (Con A) stimulation in an RWV. Splenocytes were stimulated with Con A and cultured for up to 24 h in the RWV before being allowed to "recover" under static culture conditions in the continued presence of Con A. The T-lymphocyte fraction of splenocytes was assayed during the recovery period for IL-2 secretion, expansion of the T-lymphocyte population, and expression of the activation marker CD25. Our results indicate that CD25 expression was not affected by any duration of RWV exposure. In contrast, proliferation and IL-2 secretion were inhibited by >8 and 12 h of exposure, respectively. Culture in the RWV for 24 h resulted in a near-complete loss of cellular viability during the recovery period, which was not seen in cells maintained in the RWV for 16 h or less. Taken together, these results indicate that for up to 8 h of RWV culture activation is not significantly impaired upon return to static conditions; longer duration RWV culture results in a gradual loss of activation during the recovery period most likely because of decreased T-cell viability and/or IL-2 production.     

Patterns of Species Richness at Varying Scales in Western Kenya: Planning for Agroecosystem Diversification

Agroforestry tree domestication research is geared at promoting diversification of on-farm tree species composition. A survey was conducted in western Kenya with the objective of exploring possibilities for diversification for a particular agroecosystem, involving a complete tree census, tree measurement and collection of ethnobotanical information in 201 small-scale farms. Various approaches to landscape diversification were explored, including random distribution of trees to increase alpha richness and species richness at higher scales in the landscape, and random distribution of species composition over villages to increase the average richness of villages. The results showed that random distribution would result in increments of average species richness in the landscape, without requiring increments of total and average abundance. A new, fast and exact method of calculating site-based species accumulation curves was presented. The method yielded results that were extremely close to classical algorithms using 10,000 randomisations. Four use-groups (beverage, fodder, charcoal and soil fertility enhancement) were identified as use-groups with alpha richness smaller than one species, but only beverage and fodder had lowest richness at all scales (fruit and construction wood joined the four use-groups of lowest average species richness at higher scales in the landscape). The novel approaches used in this study could be used in future biodiversity studies on species accumulation patterns, or on spatial distribution patterns of species richness in a landscape.     

The potential of adiponectin in driving arthritis

Articular adipose tissue is a ubiquitous component of human joints, but its local functions are largely unknown. Because recent studies revealed several links between adipose tissue, adipocytokines, and arthritis, we investigated the expression of the adipocytokine adiponectin and its functional role in articular adipose tissue and synovium of patients with different arthritides. In contrast to its protective role in endocrinological and vascular diseases, adiponectin was found to be involved in key pathways of inflammation and matrix degradation in the human joint. The effects of adiponectin in human synovial fibroblasts appear to be highly selective by inducing only two of the main mediators of rheumatoid arthritis pathophysiology, IL-6 and matrix metalloproteinase-1, via the p38 MAPK pathway. Owing to the observation that these effects could be inhibited by different TNF-alpha inhibitors, adipocytokines such as adiponectin may also be key targets for therapeutic strategies in inflammatory joint diseases. In summary, articular adipose tissue and adipocytokines cannot be regarded as innocent bystanders any more in chronic inflammatory diseases such as arthritis.     

Targeted irradiation of Mammalian cells using a heavy-ion microprobe

The existing focusing heavy-ion microprobe at the Gesellschaft für Schwerionenforschung in Darmstadt (Germany) has been modified to enable the targeted irradiation of single, selected cells with a defined number of ions. With this setup, ions in the range from helium to uranium with linear energy transfers (LETs) up to approximately 15,000 keV/microm can be positioned with a precision of a few micrometers in the nuclei of single cells that are growing in culture on a thin polypropylene film. To achieve this accuracy, the microbeam traverses a thin vacuum window with minimal scattering. Electron emission from that window is used for particle detection. The cells are kept in a specially designed dish that is mounted directly behind the vacuum window in a setup allowing the precise movement and the imaging of the sample with microscopic methods. The cells are located by an integrated software program that also controls the rapid deflection and switching of the beam. In this paper, the setup is described in detail together with the first experiments showing its performance. We describe the ability of the microprobe to reliably hit randomly positioned etched nuclear tracks in CR-39 with single ions as well as the ability to visualize the ion hits using immunofluorescence staining for 53BP1 as a marker of DNA damage in the targeted cell nuclei.     

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA_f^Met). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA_f^Met, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA_f^Met to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA_f^Met. This accessory factor might be unnecessary when formylated Met-tRNA_f^Met is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA_f^Met due to loss of the MIS1 gene that encodes the mitochondrial C₁-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.     

In situ pumping activity of the sponge Aplysina aerophoba, Nardo 1886

Aplysina aerophoba, Nardo 1886 is a common and well examined demosponge in the Mediterranean Sea. For sponges, as sessile, inner filter feeders, the most important function is pumping water through their canal system. This flow through is actively generated, supplies the sponge with oxygen and food particles and washes out waste products. Oxygen measurements ex situ, as a given example, show high oxygen saturations inside pumping sponges and oxygen depletion in non-pumping sponges. Thus, the oxygen situation within the sponge, like the food particle supply, waste washout and others, are directly related to its active pumping. To learn more about the poriferan function, it is important to know more about its undisturbed in situ pumping activity and consequently about the correlated conditions inside the sponge body.We conducted a tracer experiment in situ and tested sponge activity in terms of active pumping. Our technique excluded stress and disturbance to the sponges, hence minimizing experimental artefacts. The results show Aplysina aerophoba to be permanently pumping, which is implying a permanent supply of oxygen and of course food particles as well as a permanent washout of waste products and a permanent, presumably high energy consumption.We therefore conclude, that Aplysina aerophoba is always well supplied with oxygen, and that tissue anoxia or anaerobic metabolisms are of no significant importance in this sponge species. This fact of a permanent flow through in sponges will have to be taken into account for past and future hypotheses on the physiology of the sponge-microbial systems.     

Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of “Slow-Channel Syndrome” Nicotinic Receptor Mutants

The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (αS269I, βV266M, εL221F, εT264P, and εL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel–based approach to estimate the contribution of Ca²⁺ to the total cation currents, we also found that none of these mutants affects the Ca²⁺-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca²⁺-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na⁺, 1.8 mM Ca²⁺, and 1.7 mM Mg²⁺ on the extracellular side of cell-attached patches turned out be in the 5.0–9.4 pS range, representing a fractional Ca²⁺ current of ∼14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-d-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of Ca²⁺ entry into the cell. Our estimate of the rat NMDAR Ca²⁺ conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular Mg²⁺) was 7.9 pS, corresponding to a fractional Ca²⁺ current of 13%.