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Synthesis of spin-labeled nitroxyl esters of steroids.

The preparations of spin-labeled nitroxyl esters of prednisone, cortisone, deoxycorticosterone, and cholesterol are described. The ESR spectra indicate that the esters of the three steroids with an oxygen atom at the 11 position have narrower band widths than those with only protons at that position. Mass spectral, chemical, and ESR studies confirmed the structures and purity of the compounds prepared. Reaction of four esters with prednisone antibodies showed reversible binding and large crossover binding.

Animals↗

Maturation of rat spermatozoa: ESR spin labeling studies.

The ability of spermatozoa to reduce nitroxide spin--TEMPO has been used as a parameter to understand maturation, capacitation and calcium uptake of sperm obtained from Holstman strain rats. The rate of spin label reduction by sperm follows the trend--caput greater than cauda greater than corpus. With the increase in age, the electron donating capability shows first a gradual increase, for 60- to 85-day-old rats, peaking at 85 days (corresponding to puberty) and leveling off after 92 days. Calcium uptake takes place in two phases which corresponds to accumulation of and activation by calcium. The presence of polyclonal antibody which is known to cause agglutination, does not adversely affect the sperm activity.

Animals↗

The rotational diffusion of the acetylcholine receptor in Torpeda marmorata membrane fragments studied with a spin-labelled alpha-toxin: importance of the 43 000 protein(s).

The rotational diffusion of the acetylcholine (ACh) receptor in subsynaptic membrane fragments from Torpedo marmorata electric organ was investigated with a spin-labelled alpha-bungarotoxin. A toxin with two spin labels was first synthesized; the conventional electron spin resonance spectrum (e.s.r.) of this toxin bound to the receptor indicated: (1) a complete immobilization of the probes; and (2) a strong spin-spin interaction that was not, or barely, seen in solution. The modification of the degree of spin-spin interaction is taken as an indication of a toxin conformational change accompanying its binding to the ACh-receptor. To avoid spin-spin interaction a single-labelled toxin was made and used to follow the rotational diffusion of the receptor by saturation transfer e.s.r. (ST-e.s.r.). With native membranes a high immobilization of the ACh-receptor was noticed. Reduction of the membranes by dithiothreitol had little effect on this motion. Only extraction of the 43 000 protein(s) by pH 11 treatment was able to enhance the rotational diffusion of the ACh-receptor protein (rotational correlation time by ST-e.s.r. in the 0.5 - 1 X 10(-4) s range) and to allow its lateral diffusion in the plane of the membrane fragments (observed by electron microscopy after freeze-etching or negative staining).

Animals↗

Properties of the strongly immobilized signal observed in spin-labeled erythrocytes.

A strongly immobilized signal from fatty acid spin labels was observed in human erythrocytes treated with oxidizing agents such as glutaraldehyde, hydrogen peroxide, phenylhydrazine and copper-ortho-phenanthroline. This signal was also observed in freshly prepared ghosts treated with potassium superoxide and in old erythrocyte ghosts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these samples demonstrated the diffuse, nondiscrete bands of high molecular weight due to the cross-linking of membrane proteins. The temperature and pH dependences of the outer hyperfine splitting of this signal were very similar to those of bovine serum albumin. We propose that the strongly immobilized signal reflects the interaction of the lipids with the cross-linked products of membrane proteins.

Adult↗

Dynamic imaging of perfusion in human skeletal muscle during exercise with arterial spin labeling.

MR images acquired by using an arterial spin-labeling technique showed spatial and temporal variations of perfusion in the skeletal muscle of exercising humans. Perfusion measurements made during plantar flexion exercise in normal volunteers were consistent with those obtained by traditional techniques reported in the literature. Spatial heterogeneity of perfusion values clearly delineated the various muscle groups within the lower leg. These results are interpreted in terms of a quantitative model for the perfusion signal in muscle. This method can provide a useful tool in the study of muscle physiology. Magn Reson Med 42:258-267, 1999. Published 1999 Wiley-Liss, Inc.

Arteries↗

Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling.

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimer's amyloid beta (Abeta) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23-26). Despite their different aggregation properties and roles in disease, the two peptides, Abeta40 and Abeta42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.

Alzheimer Disease↗

Non-invasive measurement of perfusion: a critical review of arterial spin labelling techniques.

The non-invasive nature of arterial spin labelling (ASL) has opened a unique window into human brain function and perfusion physiology. High spatial and temporal resolution makes the technique very appealing not only for the diagnosis of vascular diseases, but also in basic neuroscience where the aim is to develop a more comprehensive picture of the physiological events accompanying neuronal activation. However, low signal-to-noise ratio and the complexity of flow quantification make ASL one of the more demanding disciplines within MRI. In this review, the theoretical background and main implementations of ASL are revisited. In particular, the perfusion quantification methods, including the problems and pitfalls involved, are thoroughly discussed in this article. Finally, a brief summary of applications is provided.

Cerebral Arteries↗

Intravascular effect in velocity-selective arterial spin labeling: the choice of inflow time and cutoff velocity.

Velocity-selective arterial spin labeling (VS-ASL) tags spins on a basis of flow velocity, instead of spatial distribution that has been commonly adopted in conventional ASL techniques. VS-ASL can potentially generate tags that are very close to the imaging plane and whereby avoid the error source of transit delay (deltat) variation independent of inflow time (TI). In practice, however, TI of VS-ASL should still be chosen with caution with respect to intravascular signal and cutoff velocity (V(c)). The presented study takes advantage of multiple TI and V(c) to systematically investigate the intravascular effect. Results demonstrate the presence of significant signal from large vessels in VS-ASL images for V(c) down to 4 cm/s. For perfusion measurement in human brain, low V(c) (<4 cm/s) is recommended. With V(c) = 2 cm/s, quantitative cerebral blood flow is 72.8 ml/100 ml/min, which is in agreement with the reported range using conventional ASL methods. In field strength of 3 T, numerical simulation shows that optimal signal-to-noise ratio efficiency can be achieved with TR/TI = 2092 ms/1664 ms for single slice and 4493 ms/1404 ms for slab imaging.

Arteries↗

Subcellular distribution of nitroxide spin-labelled 9-aminoacridine in living KB cells.

A spin-labelled derivative of 9-aminoacridine, the AATEMPO, was studied with respect to its localization in KB cells in vivo. It was found that both nuclear and mitochondrial DNAs were targets for this intercalating dye. The observed speed of intercalation and the absence of a blocked signal in cellular membranes suggested that no receptor -or carrier- proteins were implied in the penetration process. No changes in cell membrane fluidity were observed following the administration of m-AMSA, the unlabelled structural analog, to the living cells, which seemed to exclude the plasma membrane as a possible site of action of 9-aminoacridines. Side effects of phenol/chloroform mixtures and high-salt concentrations on the intercalation phenomenon were also described.

Binding Sites↗

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents.

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.

Animals↗

Understanding and optimizing the amplitude modulated control for multiple-slice continuous arterial spin labeling.

Multiple-slice perfusion imaging by continuous arterial spin labeling (CASL) is made possible by amplitude modulation (AM) of the labeling RF pulse, but perfusion sensitivity is reduced relative to the single-slice technique. A computer model of the Bloch equations for velocity driven adiabatic fast passage was developed to elucidate the compromised sensitivity to perfusion of the AM control technique for CASL. Calculations were performed over ranges of RF pulse amplitude, B1; magnetic field gradient, G; phase, phi, and frequency, f, of the modulation function; velocity, v, and relaxation times, T1 and T2, of blood. It was found that unless f>2piB1, phi determines the performance of the AM control; excessively high B1 or v reduces the efficiency of the AM control; and T1 relaxation dominates if f is too great. In vivo, in rat brain (n=5) at 2.35 T, the sensitivity of the AM technique to perfusion was 70% of the sensitivity of single-slice CASL.

Animals↗

Spin-labeled nucleotide substrates for DNA-dependent RNA polymerase from Escherichia coli.

New spin-labeled analogs of nucleoside triphosphates, 8-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenosine 5'-triphosphate ((8-AmTEMPO)ATP) and 5-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)uridine 5'-triphosphate ((5-AmTEMPO)UTP), with the probe 4-amino(2,2,6,6-tetramethylpiperidine-N-oxyl) (4-AmTEMPO) attached to C-8 of ATP and C-5 of UTP via a secondary amine bond, were synthesized in 50 and 40% yield, respectively. These analogs showed a single spot by thin layer chromatographic analysis. The absorption spectra of (8-Am-TEMPO)ATP and (5-AmTEMPO)UTP exhibit maxima at 310 and 265 nm, respectively; their X-band EPR spectra have a typical three-line pattern with lines at 3,221, 3,239, and 3,257 Gauss. The intensity ratios for mid to high field lines of the EPR derivative lines were found to be 1.03 +/- 0.02, 1.08 +/- 0.04, and 1.15 +/- 0.07 for 4-AmTEMPO, (8-AmTEMPO)ATP, and (5-AmTEMPO)UTP, respectively. The immobilization of 4-AmTEMPO bound to C-8 of ATP or bound to C-5 of UTP was observed to be 5 and 11%, respectively, as compared with free 4-AmTEMPO. The initial velocity (s-1) of [3H]UMP incorporation into RNA in the presence of [3H]UTP, CTP, GTP, and (8-AmTEMPO)ATP or ATP was measured. The percent incorporation of (8-AmTEMPO)ATP into RNA product by Escherichia coli RNA polymerase using various DNA templates is 68, 66, and 61% for pAR1435 (plasmid containing A1 promoter from T7 DNA), calf thymus DNA, and poly(dA-dT) respectively, as compared with ATP incorporation. The polymerase-catalyzed reaction of (8-AmTEMPO)ATP with (3'-OCH3)UTP yielded 5'-triphosphate delta-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenylyl (3'-5')3'-methoxy uridine in the presence of poly(dA-dT). The structure of this spin-labeled dinucleotide was identified by paper chromatographic analysis of the products of phosphodiesterase digestion. These analogs also can be used for the study by EPR spectroscopy of the dynamics of gene transcription catalyzed by RNA polymerases or of other nucleotide-utilizing enzymes.

Adenosine Triphosphate↗

Arterial spin labeling: benefits and pitfalls of high magnetic field.

Arterial spin labeling (ASL) techniques are MR imaging methods designed to measure the endogenous perfusion signal coming from arterial blood by manipulation of its magnetization. These methods are based on the subtraction of two consecutively acquired images: one acquired after preparation of the arterial blood magnetization upstream to the area of interest, and the second without any manipulation of its arterial magnetization. The subtraction of both images provides information on the perfusion of the tissue present in the slice of interest. Because ASL is a very low SNR technique, the shift from 1.5 T to 3.0 T should be regarded as a great way to increase signal-to-noise ratio (SNR). Furthermore, the concomitant increase in blood T(1) should improve the SNR of ASL further. Other effects related to poorer magnetic filed homogeneities and reduced T(2) relaxation times, however, will counterbalance both effects partially. In this article, the pros and cons of the use of ASL at high field are summarized, after a brief description of the major techniques used and their theoretical limitations. Finally, a summary of the few existing dedicated ASL perfusion techniques available are presented.

Animals↗

Kinetic analysis of fusion of hemagglutinating virus of Japan with erythrocyte membrane using spin-labeled phosphatidylcholine.

HVJ* (hemagglutinating virus of Japan containing spin-labeled phosphatidylcholine in its envelope around 10 mol %) was adsorbed onto erythrocytes or erythrocyte ghosts at various doses, and the ESR spectrum of the virus-cell system was measured at 37 degrees C. The peak-height increase for the HVJ*-ghost system was satisfactorily analyzed on the basis of envelope fusion by a first-order kinetic equation with two different rate constants. The rate constant was obtained as k1 = 0.84 min-1 and k2 = 0.011 min-1, independent of the virus dose. The fraction of virus fused at the rate constant k1 decreased with the dose. However, the average number of fast-fusing viruses per cell was nearly independent of the dose, and the value was one to two. The peak-height increase in the HVJ*-erythrocyte system was caused by both envelope fusion and phospholipid exchange catalyzed by the virus-induced hemolyzate. At lower doses, where the virus-induced hemolysis was small and, therefore, the rate of phospholipid exchange was small, the peak-height increase could be analyzed by the same kinetic equation with nearly the same rate constant value for k1 as that for HVJ*-ghosts. However, the k2 was larger than that for HVJ*-ghost, owing to the additional transfer by phospholipid exchange.

Electron Spin Resonance Spectroscopy↗

NMR observation of interactions in the combining site region of an antibody using a spin-labeled peptide antigen and NOESY difference spectroscopy.

A spin-labeled peptide antigen (TEMPOVEVPGSQHIDSQ) was used to measure NOESY difference spectra that show interactions in the binding site region of the Fab fragment of the anti-cholera toxin peptide antibody TE33. In addition to identification of peptide-Fab interactions and interactions within the bound peptide, these difference spectra show well-resolved cross peaks due to interactions within the large Fab fragment (50 kDa). These difference spectra indicate that the conformational changes in the Fab upon peptide binding are confined to the combining site region of the antibody. The NOESY difference spectra of selectively deuterated Fab molecules were used in combination with HOHAHA measurements to assign the interactions to amino acid type and to identify the interactions within the Fab as either inter- or intraresidue interactions. The assignment of interactions within the Fab to corresponding aromatic residues in the Fab sequence was facilitated by an earlier NMR-derived model calculated on the basis of NOE restraints on Fab-peptide and intra-bound-peptide distances. The new restraints on distances within the Fab, combined with the previously obtained restraints, were used to generate a refined NMR-derived model for the TE33-peptide complex.

Amino Acid Sequence↗

Antitumor and antioxidant activity of spin labeled derivatives of podophyllotoxin (GP-1) and congeners.

The spin labeled derivative of podophyllotoxin, i.e. podophyllic acid-[4-(2,2,6,6,-tetramethyl-1-piperidyloxy)] hydrazone (GP-1,2) and its congeners (GP-1-OH,3, GP-1-H, 4) were synthesized. The inhibition activity to the transplanted tumor S180 and HepA and the LD50 values of these compounds in mice were tested. Their partition coefficients and pKa values were measured. The results showed that the anticancer activity of these compounds followed the order GP-1 >GP-1-OH> GP-1-H. It can be attributed to the influence of their partition coefficients and ionization constants to the compounds properties. Meanwhile, the antilipoperoxidative activities of GP-1, GP-1-OH and GP-1-H to MDA formation of liver homogenate of rat induced by Fe2+-ascorbic acid were measured. Electrochemical studies were carried out to measure the redox midpoint potentials. The relationship between the redox midpoint potentials and the antilipoperoxidative activity followed the same order as the antitumor activity. The EPR (Electron Paramagnetic Resonance) spectroscopy of the blood from carotid artery of anesthetized Wistar rat in vivo was measured. The results indicated that there is an equilibration between GP-1 and GP-1-OH, furthermore, GP-1-H can be oxidized partly to GP-1 and equilibrated with GP-1-OH in vivo. The EPR signal intensity of GP-1 is stronger than GP-1-OH and GP-1-H. It can be concluded that the oxidative state of nitroxide in compounds play a key role to the antioxidant activity.

Animals↗

A spin label method for measuring internal volumes in liposomes or cells, applied to Ca-dependent fusion of negatively charged vesicles.

A new spin label - broadening agent system for measuring trapped volumes of vesicles or cells is described. The method seems to be more advantageous than existing procedures when volumes of highly negatively charged vesicles are to be determined. The membrane permeable spin label is TEMPONE (2,2,6,6-tetramethyl piperidone-N-oxyl), and the nonpermeable broadening agent is chromium oxalate (K3Cr(C2O4)3). Absolute values for the trapped volumes down to 0.1% in 0.1 ml can be measured with an accuracy of about +/- (1-10%). The method is used to study the final volume of fused phosphatidylserine vesicles as a function of the temperature at which the Ca-induced fusion takes place.

Animals↗

Incorporation of a ganglioside and spin-labeled ganglioside analogue into cell and liposomal membranes.

When an aqueous solution of a spin-labeled "two tail" gangliosidoid was incubated with liposomes or sheep erythrocytes, the broad single resonance line in the ESR spectrum disappeared and a signal showing an anisotropic motion appeared, indicating that the spin-labeled "two tail" gangliosidoid in the micellar state was transferred to the lipid phase of the acceptor membranes. The transfer was temperature- and time-dependent, irrespective of the acceptor membranes, indicating that the rate of transfer is determined by the escape of monomers from the micelles. The kinetics and temperature-dependence of the association of ganglioside II3NeuAc-GgOse4Cer with sheep erythrocytes was very similar to that of the "two tail" gangliosidoid, indicating that parts of ganglioside II3NeuAc-GgOse4Cer could be incorporated into the lipid phase of membranes via a similar mechanism.

Animals↗