Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31p NMR study.
(1/1206)
Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy has been used to study accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia. Lipids were extracted from rat brain homogenates and the extracts were thoroughly washed with aq. potassium ethylenediaminetetraacetic acid (EDTA). The lower organic phases were isolated and evaporated to dryness under a stream of nitrogen and the lipids were redissolved in CDCl3-CH3OH-H2O 100.0:29.9:5.2 (v/v/v) for NMR analysis. Increasing the period of post-decapitative ischemia resulted in an accumulation of two signals in the NMR spectra at 0.18 and 0.22 ppm (relative to the chemical shift of 1,2-diacyl-sn-glycero-3-phosphocholine (PCDIACYL) at -0.84 ppm). These signals were identified as originating from 1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (NAPEDIACYL) and 1-(1'-alkenyl)-2-acyl-sn -glycero-3-phospho-(N-acyl)-ethanolamine (NAPEPLAS), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N-acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs-EDTA in the preparation of the NMR samples allowed the separation of the chemical shifts of N-acyl-ethanolamine phospholipids from those of the ethanolamine phospholipids. Moreover, the chemical shift of cardiolipin was moved from 0.15 ppm observed with Cs-EDTA to about 0.31 ppm with K-EDTA. The present study demonstrates that it is possible to detect and quantify post-decapitative accumulation of NAPE subclasses (NAPEDIACYL and NAPEPLAS) in rat brains by the use of 31P NMR spectroscopy. (+info)
SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling.
(2/1206)
The exploitation of oxidative DNA lesions as biomarkers of oxidative stress in vivo requires techniques that allow for the precise and valid measurement of oxidative damage to DNA. Previously, endogenous levels of the oxidative lesion 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in rat tissues determined by a micrococcal nuclease/calf spleen phosphodiesterase-based 32P-post-labeling protocol were found to be at least 10-fold higher than those determined by HPLC with electrochemical detection. This was attributed to the adventitious oxidation of the normal nucleotides (dGp) occurring during the labeling stage of the postlabeling protocol, which could only be prevented by the introduction of additional chromatographic steps to remove the unmodified species prior to labeling. In the present study we report that an alternative snake venom phosphodiesterase-based 32P-post-labeling procedure (SVPD-postlabeling) negates the problem of adventitious oxidative damage during labeling by virtue of a unique digestion strategy. In SVPD-post-labeling, digestion yields certain lesions (thymine glycols, phosphoglycolates and abasic sites) as damage-containing dimer species which are ready substrates for labeling. In contrast, the undamaged DNA is recovered as mononucleoside species (dN) which are not substrates for labeling and so remain undetected. Furthermore, even if the mononucleosides are oxidized during labeling, they will not contribute to the level of damage detected. Indeed, we demonstrate that neither the external gamma-irradiation of the digested DNA samples nor increasing the incubation time of the labeling reaction alters the levels of damage detected by SVPD-post-labeling. The negation of adventitious oxidative effects during labeling deems that an optimized SVPD-post-labeling procedure should be well-suited for the biomonitoring of endogenous oxidative stress in vivo. (+info)
Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily.
(3/1206)
Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation. (+info)
Hypoperfusion-induced contractile failure does not require changes in cardiac energetics.
(4/1206)
Decreasing coronary perfusion causes an immediate decrease in contractile function via unknown mechanisms. It has long been suspected that this contractile dysfunction is caused by ischemia-induced changes in cardiac energetics. Our goal was to determine whether changes in cardiac energetics necessarily precede the contractile dysfunction as one would expect if a causal relationship exists. In 14 isolated rat hearts, we gradually decreased coronary perfusion using a coronary perfusate with a normal hematocrit and normal concentrations of the major metabolic substrates. Using 31P NMR spectroscopy to measure ATP, phosphocreatine (PCr), Pi, and ADP concentrations ([ATP], [PCr], [Pi], [ADP]), pH, and amount of free energy released from ATP hydrolysis (|DeltaGATP|), we found that none of these variables changed significantly until several minutes after systolic pressure had significantly decreased. Even when developed pressure had decreased by over one-third, only very slight changes in [Pi], pH, and |DeltaGATP| had occurred, with no significant changes in [ATP], [PCr], or [ADP]. Additionally, the rate of high-energy phosphate transfer between ATP and PCr did not decrease enough during hypoperfusion to explain the contractile dysfunction. We conclude that nonenergetic factors are the dominant cause of the initial decrease in systolic function when myocardial perfusion is decreased. (+info)
Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing.
(5/1206)
We have developed biodegradable fibrin-derived microbeads as potent cell carriers. The fibrin-derived microbeads, 50-200 microm in diameter, were tested for their attachment to a wide range of cell types. Fibrin-derived microbeads were shown to be greatly haptotactic to cells (such as endothelial cells, smooth muscle cells and fibroblasts), which respond to fibrinogen in contrast to keratinocytes and different cell lines derived from leukocytic lineage. The cells on fibrin-derived microbeads could be maintained for more than 10 d and achieved a high density. 31P-nuclear magnetic resonance was employed to monitor phosphate metabolism in cells, with densities on the order of 100 million cells per g of fibrin-derived microbeads. The 31P-nuclear magnetic resonance adenosine triphosphate and phosphocreatine signals, equivalent to the signal obtained with perfused normal skin, indicated that metabolism of cells on fibrin-derived microbeads was responsive to oxygenation and nutrients. Light, fluorescent, and confocal laser microscopy revealed that the porous fibrin-derived microbeads accommodate up to 200-300 cells due to their high surface area which minimized contact inhibition. Cells could degrade the fibrin-derived microbeads and be transferred to seed culture flasks without trypsinization. In a pig skin wound healing model, fibrin-derived microbeads + fibroblasts were transplanted into full thickness punch wounds. This procedure was compared with other treatment modalities, such as the addition of human platelet-derived growth factor BB or fibrin-derived microbeads alone. By the third day after wounding, only the wounds in which fibroblasts on fibrin-derived microbeads were added showed significant formation of granulation tissue. Based on the above, we project many uses of our novel fibrin-derived microbead technology for cell culturing, wound healing and tissue engineering. (+info)
Evaluation of the effects of photodynamic therapy with phosphorus 31 magnetic resonance spectroscopy.
(6/1206)
Magnetic resonance spectroscopy in situ was used to study changes in phosphorus 31 metabolism after photodynamic therapy (PDT) of transplanted HeLa cell tumours. Tumours were irradiated 2 h after administration of ATX-S10 (8-formyloximethylidene-7-hydroxy-3-ethenyl-2,7,12,18, tetramethyl-porphyrin-13,17-bispropionil aspartate), a new photosensitizer and chlorin derivative. Nuclear magnetic resonance spectra were measured prior to illumination and 1, 3, 7, 14, 21 and 28 days after PDT on each mouse. A drastic decrease in adenosine triphosphate (ATP) and a concomitant increase in inorganic phosphate (Pi) were evident on the first day after PDT in all cases. The beta-ATP/total phosphate (P) ratio was 0.64 +/- 0.29% (average +/- s.d.) in complete response, 0.67 +/- 0.30% in recurrence and 2.45 +/- 0.93% in partial response. Comparison of this ratio to the histological findings revealed that the beta-ATP/total P ratio reflects the HeLa cell tumours which survived PDT. In other words, partial response on the one hand was distinguished from complete response and recurrence on the other with this ratio 1 day after PDT (P < 0.05). In addition, the ratio of phosphomonoester (PME) to Pi rose beyond 1.0 when macroscopic recurrence occurred, while it stayed under 1.0 in complete response. This finding suggests that the recurrence of HeLa cell tumours can be detected by the PME/Pi ratio. (+info)
Effect of an endothelin receptor antagonist and an angiotensin converting enzyme inhibitor on metabolism and contraction in the ischemic and reperfused rabbit heart.
(7/1206)
The effect of an endothelin (ET) A/ETB receptor antagonist, TAK-044, and/or an angiotensin converting enzyme (ACE) inhibitor, temocaprilat, on myocardial metabolism and contraction during ischemia and reperfusion was examined by phosphorus 31-nuclear magnetic resonance (31P-NMR) in Langendorff rabbit hearts. After normothermic 15 min global ischemia, 60min of postischemic reperfusion was carried out. TAK-044 and/or temocaprilat was administered from 40 min prior to the global ischemia. Adenosine triphosphate (ATP), creatine phosphate, inorganic phosphate, pH, left ventricular systolic developed pressure (LVDev.P), left ventricular end-diastolic pressure (LVEDP) and coronary flow were measured. Twenty-eight hearts were divided into 4 experimental groups consisted of seven hearts each: Group I consisted of controls, Group II was perfused with TAK-044 (10(-6) mol/L), Group III was perfused with temocaprilat (10(-6) mol/L), and Group IV was perfused with TAK-044 (10(-6) mol/L) in combination with temocaprilat (10(-6) mol/L). Group II showed a more early recovery of ATP during postischemic reperfusion (82+/-3%) compared with Group I (71+/-3%). Group III showed a significant inhibition of the decrease in ATP during global ischemia (54+/-3%) compared with Group I (45+/-3%). Group IV also showed a significant marked inhibition of the decrease in ATP during global ischemia (59+/-5%) and a more significant improvement on recovery of ATP during postischemic reperfusion (86+/-3%) compared with the other 3 groups. There were no differences in LVDev.P, LVEDP and coronary flow among these groups. In conclusion, TAK-044 in combination with temocaprilat had a significant potentiation on myocardial metabolism during both ischemia and reperfusion. (+info)
Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes.
(8/1206)
Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment. (+info)