Comparison of perturbation effect of propranolol, verapamil, chlorpromazine and carbisocaine on lecithin liposomes and brain total lipid liposomes. An EPR spectroscopy study.

Effect of verapamil, propranolol, chlorpromazine and carbisocaine on dynamics and/or order of liposomes (perturbation effect), prepared from different molar ratios of lecithin (PC) and rat brain total lipids (TL) was studied by EPR spectroscopy using spin probes 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The PC liposomes had higher dynamics and/or lower order than the TL liposomes. The perturbation effect of the drugs depended largely on the lipid composition of the liposomes. The drugs at the drug/lipid molar ratios from 0.1 to 1 increased membrane dynamics and/or decreased membrane order. The drugs had the most pronounced perturbation effect in the liposomes prepared from brain total lipids. The effect of the drugs decreased with decreasing the TL/PC ratio in the liposomes and was lowest, almost diminished, in the PC liposomes. Increasing concentration of the drugs decreased the difference between the dynamics and/or order of the PC and TL liposomes and so eliminated the influence of lipid composition on these membrane parameters. The results emphasize the role of lipid composition in studies concerning drug-lipid interactions in model and biological membranes.     

Perturbation effect of eight calcium channel blockers on liposomal membranes prepared from rat brain total lipids.

EPR spectroscopy of phosphatidylcholine or stearic acid labeled at the doxyl group at the 16-carbon position was used to compare the perturbation effect of eight calcium channel blockers (CB) on overall dynamics/disorder of the hydrophobic part of liposome membranes prepared from rat brain total lipids at the drug/lipid molar ratio of 1/2. Nifedipine (NIF), nimodipine, niludipine and nitrendipine had a minor effect on the dynamics/disorder of the liposome membranes, whereas the disordering effect of verapamil (VER), mepamil, gallopamil and diltiazem was more pronounced. Concentration dependence of the overall disordering effect of VER on liposomal membranes was found at the VER/lipid ratio greater than 0.02 and for the tranquilizer thioridazine greater than 0.005. VER exerted a disordering effect at the hydrophobic part of synaptosomal membranes at concentrations greater than or equal to 0.32 mmol/l, whereas NIF did not exhibit a disordering effect even at concentrations of 10-20 mmol/l.     

Protein Dynamics Associated with Failed and Rescued Learning in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (Hsa21). Although it is the most common genetic cause of intellectual disability (ID), there are, as yet, no effective pharmacotherapies. The Ts65Dn mouse model of DS is trisomic for orthologs of ∼55% of Hsa21 classical protein coding genes. These mice display many features relevant to those seen in DS, including deficits in learning and memory (L/M) tasks requiring a functional hippocampus. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist, memantine, was shown to rescue performance of the Ts65Dn in several L/M tasks. These studies, however, have not been accompanied by molecular analyses. In previous work, we described changes in protein expression induced in hippocampus and cortex in control mice after exposure to context fear conditioning (CFC), with and without memantine treatment. Here, we extend this analysis to Ts65Dn mice, measuring levels of 85 proteins/protein modifications, including components of MAP kinase and MTOR pathways, and subunits of NMDA receptors, in cortex and hippocampus of Ts65Dn mice after failed learning in CFC and after learning was rescued by memantine. We show that, compared with wild type littermate controls, (i) of the dynamic responses seen in control mice in normal learning, >40% also occur in Ts65Dn in failed learning or are compensated by baseline abnormalities, and thus are considered necessary but not sufficient for successful learning, and (ii) treatment with memantine does not in general normalize the initial protein levels but instead induces direct and indirect responses in approximately half the proteins measured and results in normalization of the endpoint protein levels. Together, these datasets provide a first view of the complexities associated with pharmacological rescue of learning in the Ts65Dn. Extending such studies to additional drugs and mouse models of DS will aid in identifying pharmacotherapies for effective clinical trials.     

The potential pitfalls of using 1,1-diphenyl-2-picrylhydrazyl to characterize antioxidants in mixed water solvents

Approaching living systems, aqueous solutions are appropriate to characterize antioxidants, whereas the frequently used standard 1,1-diphenyl-2-picrylhydrazyl (DPPH) is insoluble in water. Therefore, mixed water-ethanol solvents were investigated using the electron paramagnetic resonance (EPR) spectroscopy. Two forms of DPPH were identified: at higher ethanol ratios a quintet spectrum characteristic of solutions, and at lower ratios, a singlet spectrum typical for solid DPPH, were found. Mixed solvents with 0-50% (v/v) water reproduced the same antioxidant equivalent points well and the reaction rate between DPPH and the antioxidant may increase considerably with increasing water ratios, as demonstrated using vitamin E as an antioxidant. But at still higher water ratios (70-90% (v/v)) the antioxidant activities dropped, since a part of the DPPH in the aggregated form does not react sufficiently with the antioxidants. Characteristics of the most common antioxidants were determined in ethanol or its 50% (v/v) aqueous solution.     

Portage connectivity does not predict establishment success of canoe-mediated dispersal for crustacean zooplankton

Although community structure may be largely determined by local abiotic and biotic conditions under moderate levels of dispersal, anthropogenic activities can enhance dispersal rates far beyond what would otherwise occur in natural systems. We investigated the potential impact of recreational canoeing on crustacean zooplankton community structure in Killarney Provincial Park, Canada, where canoes that are transported between lakes via portage routes may enhance zooplankton community connectivity by providing a dispersal “short-cut.” We conducted a study to (1) quantify zooplankton attachment to canoe hulls after paddling through a lake and assess the importance of canoes to overall seasonal dispersal within a lake relative to other means of dispersal, (2) test the prediction that zooplankton survivorship is negatively correlated with portage duration using a mesocosm experiment, and (3) test whether variation in lake community composition was better explained by models based on reduced portage-corrected distances or true edge-to-edge distances between lakes along popular canoe routes. Here, we report the findings that canoes have the potential to act as frequent dispersal vectors, but appear to have little impact on community structure in portage-connected lakes. Substantial numbers of adult zooplankton became attached to canoe hulls and were able to establish viable populations even after exposure to portage conditions for 30 min. However, canoe-mediated dispersal only accounted for a very small proportion (<1% in this case) of overall seasonal dispersal. Moreover, environmental variables explained the greatest amount of variation in community composition among park lakes. Nevertheless, this study indicates that canoe dispersal could be more effective for specific species such as Sida crystallina than is evident by analysis of entire communities and could facilitate the spread of invasive species amenable to attaching to boat hulls. Thus, the debate about whether community composition is more strongly influenced by local environmental conditions or regional dispersal may vary depending on the scale of consideration (i.e., individual species vs. whole community).     

Comparison of dynamics of lecithin liposomes and brain total lipid liposomes with synaptosomal membranes. An EPR spectroscopy study.

Dynamics and/or order of the hydrophobic part of phosphatidylcholine (PC) liposomes and rat brain total lipid (TL) liposomes and synaptosomes were studied and compared by EPR spectroscopy using the spin probes 5 or 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The dynamics and/or order of the hydrophobic part of TL liposomes or synaptosomes were similar but differed largely from those of PC liposomes. The dynamics of the hydrophobic part of the liposomes decreased gradually with the increasing TL/PC ratio in the sample. To obtain in TL liposomes or synaptosomes the same EPR spectrum parameters as in PC liposomes at 37 degrees C, the formers have to be heated to temperatures of approximately 50-60 degrees C. The dynamics and/or order of the hydrophobic part of lecithin liposomes at 5-10 degrees C were comparable with those of TL liposomes or synaptosomes at 37 degrees C. The results emphasize the role of the lipid composition in studies concerning drug-lipid and protein-lipid interactions in model and biological membranes.     

Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers

Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.     

Photodegradation of hyaluronic acid: EPR and size exclusion chromatography study.

Photochemically induced radical reactions involving the lateral sequences and the end macromolecular chain groups of hyaluronic acid in aqueous solutions at 293K were studied by EPR spin trapping technique with DMPO (5,5-dimethylpyrroline-1-oxide). In the first 1-10 minutes of irradiation EPR indicates spin adducts of two carbon centered radicals with the splitting constants of aN = 1.60 mT, aH = 2.51 mT and aN = 1.56 mT, aH = 2.28 mT. After longer irradiation time (over 10 minutes) dominate two further DMPO adducts of radicals centered on hetero-atoms with splitting constants of aN = 1.44 mT, aH = 1.60 mT and of aN = 1.49 mT, aH = 1.49 mT. Simultaneously, molecular weight followed by SEC decreases, suggesting that UV irradiation leads to the breaking of interglycosidic bonds of hyaluronic acid main macromolecular chain.     

Effects of water clarity and other environmental factors on trophic niches of two sympatric piscivores

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Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus)

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.