Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine

Nifedipine, verapamil and stobadine were tested and compared with butylated hydroxytoluene (BHT) as possible free radical scavengers inhibiting lipid peroxidation in phosphatidylcholine liposomes. Liposomes were peroxidized by incubation in air at 50 degrees C. Verapamil less than nifedipine less than BHT less than stobadine depressed the lipid peroxidation as detected spectroscopically for conjugate diene and thiobarbituric acid product formation. Verapamil and stobadine were tested as OH radical scavengers in a Fenton-type reaction against spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO), as detected by ESR spectroscopy. The tested drugs competed with DMPO in trapping OH radicals, with stobadine being more effective than verapamil. ESR spectra of nifedipine in the incubated liposomes revealed that nifedipine could be involved in free radical reactions in the liposomes leading to nifedipine-stable radical(s) which were immobilized in the membrane. The obtained results suggest that some of the beneficial effects of the studied drugs can be mediated in disease by their ability to scavenge free radicals and by their protective effect on lipid peroxidation.     

Comparison of the potency of five potential beta-adrenoceptor blocking drugs and eight calcium channel blockers to inhibit platelet aggregation and to perturb liposomal membranes prepared from platelet lipids.

Five potential beta-adrenoceptor blocking (BAB) compounds, alkylesters of 4-[(2-hydroxy-3-alkylamino)propoxy] phenylcarbamic acid, and eight calcium channel blockers (CB), i.e. nifedipine, nimodipine, niludipine, nitrendipine, verapamil, gallopamil, mepamil and diltiazem, were compared as to their inhibitory effect on thrombin induced aggregation of washed rat platelets and their effect on dynamics/disorder of liposomal membranes prepared from platelet lipids, studied by EPR spectroscopy of a lipid spin probe. The anti-aggregatory potency of the BAB and CB drugs was effective within the concentration range of 0.01-1 mmol/l. The antiaggregatory potency of BAB increased in the order BL-143 < BL-243 < BL-343 < BL-443 < BL-543 and among the CB, nifedipine and diltiazem were least potent, whereas nitrendipine and mepamil were the most potent drugs. The potency of the other CB tested was intermediate. The BAB drugs increased the dynamics/disorder of the liposomes in the same order as they inhibited platelet aggregation, whereas there was no relationship between antiaggregatory effect of CB and their influence on dynamics/disorder of the liposomes. Nifedipine, nimodipine, niludipine and nitrendipine had a minor perturbation effect on the liposomes, whereas verapamil, mepamil, gallopamil and diltiazem pronouncedly increased the dynamics/disorder of the hydrophobic part of the liposomes. The results indicate that the anti-aggregatory activity of BAB drugs may be mediated, at least partially, through their perturbation effect on the lipid part of biological membranes.     

Trophic variability of Arctic fishes in the Canadian Beaufort Sea: a fatty acids and stable isotopes approach

Trophic ecology of most demersal Arctic fishes remains one of the major knowledge gaps for understanding food web dynamics and connectivity among ecosystems. In this study, fatty acids (FA) and stable isotopes (SI) were used to study the feeding ecology of seven species (n = 106) of the most abundant benthic fishes (eelpouts, sculpins and agonids) in the Canadian Beaufort Sea from shallow (20–75 m), slope (200–350 m) and deep (500–1000 m) habitats. Both FA and SI results revealed among- and within-species variability in diet composition. Correspondence analysis of FA signatures identified high within-species variability in diet, resulting in high overlap among species. Calanus-derived FA were present in all species (Calanus markers up to 13 % of total FA) and were particularly important in Ribbed Sculpin, Adolf’s and Longear Eelpout collected in deep habitats, suggesting a strong contribution of pelagic-derived FA to benthic fish communities. Incorporation of this signal in the benthos may result from either direct consumption of deep overwintering copepods (i.e., off-bottom feeding) or through detrital accumulation in benthic invertebrate prey. Mean SI values differed among species and indicated that a large range of trophic positions (δ¹⁵N varied from 14.09 to 17.71 ‰ for Canadian Eelpout and Adolf’s Eelpout, respectively) and carbon dietary sources are preyed upon (δ¹³C range from ?21.13 to ?23.85 ‰ for Longear Eelpout and Ribbed Sculpin, respectively). SI analyses suggested that most species examined were low- to mid-trophic generalist benthic carnivores, with the exception of Ribbed Sculpin, which was a low-trophic pelagic predator.     

EPR spectroscopy of free radical intermediates of antiarrhythmic-antihypoxic drug stobadine, a pyridoindole derivative

Mechanisms of antioxidant action of stobadine, a pyridoindole derivative with cardioprotective and antihypoxic properties, has been probed using EPR spectroscopy. Oxidation of stobadine by PbO2/tBuOOH in benzene results in the formation of nitroxide radical observable directly by EPR spectroscopy at room temperature, indicating conversion of indolic amino group to the corresponding nitroxide.     

Influence of glutathione and its derivatives on fibrin polymerization

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with Aalpha251 recombinant fibrinogen (lacking alphaC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the alphaC region of fibrinogen.     

S-nitrosothiol-modified dendrimers as nitric oxide delivery vehicles

The synthesis and characterization of two generation-4 polyamidoamine (PAMAM) dendrimers with S-nitrosothiol exteriors are reported. The hyperbranched macromolecules were modified with either N-acetyl-D, L-penicillamine (NAP) or N-acetyl-L-cysteine (NACys) and analyzed via 1H and 13C NMR, UV absorption spectroscopy, MALDI-TOF mass spectrometry, and size exclusion chromatography. Treatment of the dendritic thiols with nitrite solutions yielded the corresponding S-nitrosothiol nitric oxide (NO) donors (G4-SNAP, G4-NACysNO). Chemiluminescent NO detection demonstrated that the dendrimers were capable of storing approximately 2 micromol NO x mg (-1) when exposed to triggers of S-nitrosothiol decomposition (e.g., light and copper). The kinetics of NO release were found to be highly dependent on the structure of the nitrosothiol (i.e., tertiary vs primary) and exhibited similar NO release characteristics to classical small molecule nitrosothiols reported in the literature. As a demonstration of utility, the ability of G4-SNAP to inhibit thrombin-mediated platelet aggregation was assayed. At equivalent nitrosothiol concentrations (25 microM), the G4-SNAP dendrimer resulted in a 62% inhibition of platelet aggregation, compared to only 17% for the small molecule NO donor. The multivalent NO storage, the dendritic effects exerted on nitrosothiol stability and reactivity, and the utility of dendrimers as drug delivery vehicles highlight the potential of these constructs as clinically useful S-nitrosothiol-based therapeutics.     

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.     

Antioxidant properties of tea investigated by EPR spectroscopy

The antioxidant properties of green, black and mixed (fruit) tea samples of different origin were investigated by means of EPR spectroscopy. A six line EPR spectrum of solid tea samples indicates the presence of Mn(II) ions and it is superimposed with a sharp singlet line attributed to semiquinone radical species (Delta H(pp)=1 mT; g=2.0022). Antioxidant properties of aqueous tea extracts in H(2)O(2)/NaOH/dimethylsulfoxide system generating reactive radicals (*OH, O(2)*-), *CH(3)) were followed by spin trapping technique. In addition, antioxidant capacity of these samples was assessed using stable radicals 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL). Typically, the highest antioxidant potential to terminate superoxide radicals was found in green teas, followed by black and fruity teas. The pro-oxidant activity of green teas evidenced by spin traps was promoted in samples with higher Mn(II) and ascorbic acid concentrations. Various sources of free radicals used in the antioxidant tests due to their specific action show different termination rates in the presence of the individual tea samples.     

An intensive hands-on course designed to teach molecular biology techniques to physiology graduate students

To address a growing need to make research trainees in physiology comfortable with the tools of molecular biology, we have developed a laboratory-intensive course designed for graduate students. This course is offered to a small group of students over a three-week period and is organized such that comprehensive background lectures are coupled with extensive hands-on experience. The course is divided into seven modules, each organized by a faculty member who has particular expertise in the area covered by that module. The modules focus on basic methods such as cDNA subcloning, sequencing, gene transfer, polymerase chain reaction, and protein and RNA expression analysis. Each module begins with a lecture that introduces the technique in detail by providing a historical perspective, describing both the uses and limitations of that technique, and comparing the method with others that yield similar information. Most of the lectures are followed by a laboratory session during which students follow protocols that were carefully designed to avoid pitfalls. Throughout these laboratory sessions, students are given an appreciation of the importance of proper technique and accuracy. Communication among the students, faculty, and the assistant coordinator is focused on when and why each procedure would be used, the importance of each step in the procedure, and approaches to troubleshooting. The course ends with an exam that is designed to test the students' general understanding of each module and their ability to apply the various techniques to physiological questions.     

Editing of the serotonin 2C receptor pre-mRNA: Effects of the Morris Water Maze

The pre-mRNA encoding the serotonin 2C receptor, HTR2C (official mouse gene symbol, Htr2c), is subject to adenosine deamination that produces inosine at five sites within the coding region. Combinations of this site-specific A-to-I editing can produce 32 different mRNA sequences encoding 24 different protein isoforms with differing biochemical and pharmacological properties. Studies in humans have reported abnormalities in patterns of HTR2C editing in psychiatric disorders, and studies in rodents show altered patterns of editing in response to drug treatments and stressful situations. To further explore the biological significance of editing of the Htr2c mRNA and its regulation, we have examined patterns of Htr2c editing in C57BL/6J mice after exposure to the hidden platform version of the Morris Water Maze, a test of spatial learning that, in mice, is also associated with stress. In brains of both swimming controls and mice trained to find the platform, subtle time dependent changes in editing patterns are seen as soon as 1 h after a probe trial and typically last less than 24 h. Changes in whole brain with cerebellum removed differ from those seen in isolated hippocampus and cortex. Unexpectedly, in hippocampi from subsets of mice, abnormally low levels of editing were seen that were not correlated with behavior or with editing levels in cortex. These data implicate responses to spatial learning and stress, in addition to stochastic processes, in the generation of subtle changes in editing patterns of Htr2c.