Long-term preservation of active luminous bacteria by lyophilization

A simple method for long-term preservation of luminous bacteria is described. Cells of Vibrio fischeri, Photobacterium leiognathi and four strains of P. phosphoreum were suspended in a protective medium of low ionic strength (1% NaCI) supplemented with 15% lactose and 2% soluble starch, and lyophilized. The freeze-dried preparations were sealed under vacuum and stored at 4°C. Luminous bacteria were resuscitated affer six months by adding 2% NaCl up to the original volume. The rehydrated cells exhibited 16-28% of initial bioluminescence so that they could be used for a microbial test of toxicity (the Microtox test). This method is also useful for maintaining luminous bacteria in strain collections.     

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated,Low-Density Plasma Membrane Fragments

Principal Findings

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C³⁵¹I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025–0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [³²P]GTPase and [³⁵S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound G_i1α within δ-opioid receptor-G_i1α fusion protein, but it was also valid for PTX-sensitive G proteins of G_i/G_o family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the “wobble in cone” model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.

Summary

Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.     

Exposure to 17β-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida

Background

The environmental impacts of various substances on all levels of organisms are under investigation. Among these substances, endocrine-disrupting compounds (EDCs) present a threat, although the environmental significance of these compounds remains largely unknown. To shed some light on this field, we assessed the effects of 17β-oestradiol on the growth, reproduction and formation of free radicals in Eisenia fetida.

Methodology/Principal Findings

Although the observed effects on growth and survival were relatively weak, a strong impact on reproduction was observed (50.70% inhibition in 100 μg/kg of E₂). We further demonstrated that the exposure of the earthworm Eisenia fetida to a contaminant of emerging concern, 17β-oestradiol (E₂), significantly affected the molecules involved in antioxidant defence. Exposure to E₂ results in the production of reactive oxygen species (ROS) and the stimulation of antioxidant systems (metallothionein and reduced oxidized glutathione ratio) but not phytochelatins at both the mRNA and translated protein levels. Matrix-assisted laser desorption/ionization (MALDI)-imaging revealed the subcuticular bioaccumulation of oestradiol-3,4-quinone, altering the levels of local antioxidants in a time-dependent manner.

Conclusions/Significance

The present study illustrates that although most invertebrates do not possess oestrogen receptors, these organisms can be affected by oestrogen hormones, likely reflecting free diffusion into the cellular microenvironment with subsequent degradation to molecules that undergo redox cycling, producing ROS, thereby increasing environmental contamination that also perilously affects keystone animals, forming lower trophic levels.     

Mesenchymal stromal cells prolong the lifespan in a rat model of amyotrophic lateral sclerosis

Background aimsAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals’ lifespan.MethodsAt the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fluorescent protein (GFP⁺ MSC) on the same day, intraspinally (10⁵ cells) and intravenously (2 × 10⁶ cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis.ResultsThe combined grafting of MSC into symptomatic rats had a significant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 ± 3.33 days compared with 179 ± 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFP⁺ MSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site.ConclusionsIntraspinal and intravenous transplantation of MSC has a beneficial and possibly synergistic effect on the lifespan of ALS animals.     

Genetic differences within natural and planted stands of <Emphasis Type="Italic">Quercus petraea</Emphasis>

Five sessile oak [Quercus petraea (Matt.) Liebl.] stands from the Czech Republic were studied to learn about the impact of different types of forest management regimes on the genetic differences among tree populations and on population structures. One population had not been markedly affected by human activity, two populations represented unplanted stands that were extensively managed for a long period of time using the coppice system, and two populations were planted stands. Approximately 100 trees from each stand were mapped and subsequently genotyped using 10 nuclear microsatellite loci. We determined the spatial genetic structure of each population and the genetic differentiation among the populations. We found that: (i) the populations were genetically differentiated, but the differences between the unplanted and planted stands were not markedly significant; (ii) the genetic differentiation among the populations depended on the geographical distribution of the populations; (iii) within unplanted stands, a strong spatial genetic structure was seen; and (iv) within planted stands, no spatial genetic structure was observed. Our findings implies that the analysis of spatial genetic structure of the sessile oak forest stand can help reveal and determine its origin.     

The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent toxoplasmosis: implications for the behavioral manipulation hypothesis

Background

The highly prevalent parasite Toxoplasma gondii reportedly manipulates rodent behavior to enhance the likelihood of transmission to its definitive cat host. The proximate mechanisms underlying this adaptive manipulation remain largely unclear, though a growing body of evidence suggests that the parasite-entrained dysregulation of dopamine metabolism plays a central role. Paradoxically, the distribution of the parasite in the brain has received only scant attention.

Methodology/Principal Findings

The distributions of T. gondii cysts and histopathological lesions in the brains of CD1 mice with latent toxoplasmosis were analyzed using standard histological techniques. Mice were infected per orally with 10 tissue cysts of the avirulent HIF strain of T. gondii at six months of age and examined 18 weeks later. The cysts were distributed throughout the brain and selective tropism of the parasite toward a particular functional system was not observed. Importantly, the cysts were not preferentially associated with the dopaminergic system and absent from the hypothalamic defensive system. The striking interindividual differences in the total parasite load and cyst distribution indicate a probabilistic nature of brain infestation. Still, some brain regions were consistently more infected than others. These included the olfactory bulb, the entorhinal, somatosensory, motor and orbital, frontal association and visual cortices, and, importantly, the hippocampus and the amygdala. By contrast, a consistently low incidence of tissue cysts was recorded in the cerebellum, the pontine nuclei, the caudate putamen and virtually all compact masses of myelinated axons. Numerous perivascular and leptomeningeal infiltrations of inflammatory cells were observed, but they were not associated with intracellular cysts.

Conclusion/Significance

The observed pattern of T. gondii distribution stems from uneven brain colonization during acute infection and explains numerous behavioral abnormalities observed in the chronically infected rodents. Thus, the parasite can effectively change behavioral phenotype of infected hosts despite the absence of well targeted tropism.