An electron paramagnetic resonance study of skeletal muscle membrane fluidity in malignant hyperthermia

Skeletal muscle sarcolemma (SL), transverse tubule (TT) and heavy sarcoplasmic reticulum (HSR) membranes were isolated from malignant hyperthermia susceptible (MHS) and normal pigs, and the rotational dynamics of lipid hydrocarbon chain motion was examined by electron paramagnetic resonance (EPR) spectroscopy. The stearic acid spin probe 16-SASL was incorporated into MHS and normal membranes and both the order parameter (S) and effective correlation time (tau r) of probe motion were calculated from spectra recorded over the temperature range of 2 to 40 degrees C. At any given temperature, TT membranes exhibited significantly greater values for both the S and tau r of probe motion than did SL, which exhibited significantly greater values than did HSR membranes. The order of decreasing S and tau r values for 16-SASL mobility correlated with the decreasing cholesterol content of these membranes (TT greater than SL greater than HSR), however there was no difference in the S or tau r values for a given membrane fraction isolated from both MHS and normal muscle. Arrhenius plots of 16-SASL mobility in SL, TT and HSR were linear from 2 to 40 degrees C, indicating no abrupt thermotropic change in the lipid hydrocarbon phase of any of the membrane types studied. Apparent activation energies (Ea), calculated from the Arrhenius plots, were similar for MHS and normal membranes derived from a given cellular location. However, the Ea of probe motion for TT membranes (2.3 +/- 0.1 and 2.4 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) was significantly less than for SL (3.4 +/- 0.4 and 2.9 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) which, in turn, was significantly less than the Ea for HSR (3.7 +/- 0.1 and 3.7 +/- 0.1 kcal/mol/degree for MHS and normal, respectively). Since 16-SASL motion was similar in MHS and normal membranes, we conclude that there is no evidence for a generalized membrane defect affecting lipid mobility in these MHS muscle membranes.     

A novel small molecule HSP90 inhibitor,NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1^G93A similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca²⁺ accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1^G93A transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo.     

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase,and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5′-nucleotidase (5′-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5′-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5′-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.     

Global diversity of springtails (Collembola; Hexapoda) in freshwater

Water-dependency appeared independently in several clades of the class Collembola, which is basically of terrestrial origin according to recent phylogenetic analyses. Though moderately diversified (less than 8,000 species), Collembola are among the most numerous terrestrial arthropods in wetland communities, with a small number of species living on the surface of water. Many species are dependent on water-saturated atmosphere of caves, and on snow or ice in high mountains. A total of 525 water-dependent species have been recognized, of which 103 are linked to free freshwaters and 109 to anchialine or marine waters. Many interstitial species are also dependent to an unknown extent on water saturation in the deep layers of the soil. The numbers provided here are underestimates, as Collembola are extremely poorly known outside the Holarctis, and the ecology of described species usually not documented. However, a general biogeographical pattern is emerging from available data. The most remarkable feature is that about 15% of the fauna is water-dependent in the holarctic region, compared to 4% in the tropics and southern hemisphere. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

A Chemically Defined Medium for Rabbit Embryo Cryopreservation

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbecco''s phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v⁻¹) of CRYO3 or 18% (v.v⁻¹) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.