Biomedical subjects
Publications and source records attributed to C C Hanstock.
The modification of the RF field distribution of a surface coil, which is brought about by both loading and skin effect conditions arising from the proximity of weakly conducting saline samples, is demonstrated. A procedure for the calibration of the depth of the spectral acquisition region in depth-pulse localized in vivo spectroscopy is shown to be acceptable.
Spectral editing techniques and localization of 1H signals were applied to monitor lactate accumulation in a circumscribed region of brain damage. The experiments were performed at 2.35 T (100 MHz) in a 40-cm bore magnet. Following unilateral craniectomy in anesthetized adult cats, a two-turn surface coil was positioned over the dural surface. Proton spectra were obtained before and 1-5 h after production of a cortical cold lesion from three curved shells of brain tissue, each approximately 3 mm thick. The localized spectrum was obtained from each region with and without spectral difference editing for the lactate CH3 protons, but always with the maximum excitation produced by the semiselective binomial pulse centered on the lactate CH3 resonance. Region 1 represented the damaged area, Region 2 was located immediately below Region 1, and Region 3 was immediately below Region 2. Spin-echo magnetic resonance imaging was used to confirm the relationship between the location of the lesion and the regions from which the spectra were obtained. Spectra obtained without lactate editing showed, in addition to the N-acetylaspartate peak, a large lactate peak in Region 1 after production of the cold lesion. In Regions 2 and 3, changes in lactate were more difficult to assess owing to the presence of a lipid peak at a similar frequency that results from incomplete suppression by the spin-echo pulse sequence alone. Spectra acquired using lactate editing did not contain the lipid peak and clearly showed relatively small lactate accumulations in Regions 2 and 3.(ABSTRACT TRUNCATED AT 250 WORDS)
By incorporating the hyperbolic secant inversion pulses with the image-selected in vivo spectroscopy localization technique and by applying a gradient-echo imaging method, we have selected only the 31P NMR signals from orthotopic human kidney and liver, using a single concentric 1H/31P surface coil. Corresponding to the experimental results on animal studies, the phosphocreatine signal is dramatically reduced in the localized spectra. Our localization strategy also allows us to shim easily on the well-defined volume of interest and leads to high-resolution spectra that exhibit multiplet structure. Our results indicate that we can obtain localized signals from deep small organs and point the way for other human metabolism studies.
Using a surface coil, we have obtained 1H NMR spectra from metabolites in the human brain. Localization was achieved by combining depth pulses with image-selected in vivo spectroscopy magnetic field gradient methods. 1H spectra in which total creatine (3.03 ppm) has a signal/noise ratio of 95:1 were obtained in 4 min from 14 ml of brain. A resonance at 2.02 ppm consisting predominantly of N-acetylaspartate was measured relative to the creatine peak in gray and white matter, and the ratio was lower in the white matter. The spin-spin relaxation times of N-acetylaspartate and creatine were measured in white and gray matter and while creatine relaxation times were the same in both, the N-acetylaspartate relaxation time was longer in white matter. Lactate was detected in the normoxic brain and the average of three measurements was approximately equal to 0.5 mM from comparison with the creatine plus phosphocreatine peak, which was assumed to be 10.5 mM.
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Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.
The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete 1H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50 degrees C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the 1H-NMR experiment was established independently of the NMR evidence by 32P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete 1H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.
The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20 degrees C was confirmed by temperature variable 1H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T1 experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.
The two deoxyribonucleotides [d(CpGpApTpCpG)]2 and [d(CpGpCpG)]2 were synthesized by the phosphotriester method. Their duplex form under the conditions of the 1H-nmr experiments was proven by end 32P labeling with T4 polynucleotide kinase followed by butt end joining employing the absolute specificity of T4 ligase for double stranded DNA and analysis using gel electrophoresis and autoradiography. Complete nmr assignment of the 1H chemical shifts and coupling constants was achieved. The assignments were secured using sequential decoupling, NOE difference measurements, and two-dimensional COSY and SECSY experiments. Spectrum simulation confirmed the experimental values of chemical shifts and coupling constants. The techniques for the assignment outlined together with 31P and 2-D heteronuclear shift correlation permit an approach to a systematic analysis of more complex single-strand and duplex oligodeoxyribonucleotides.
The interactions of the low cardiotoxic antitumor agents 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9, 10-anthracenedione (mitoxantrone) and 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazoyl-2-yl)hydrazone] (bisantrene) with pBR322 and PM2 DNA have been examined by electron microscopy. Direct evidence was obtained for intercalative binding of both drugs, with mitoxantrone causing a 13% average length increase in pBR322 corresponding to approximately 580 drug molecules per circle at saturation and bisantrene causing an 11% increase in length corresponding to approximately 480 drug molecules bound per circle. Considerations of the known GC preference for non-nearest neighbor binding of the drugs and inspection of the known sequence of pBR322 suggest that the available intercalation sites are occupied and that additional external electrostatic binding of the cationic drugs also occurs. An apparent difference in behavior of mitoxantrone as compared with that of bisantrene in causing no net increase in length of supercoiled pBR322 was shown to be attributable to an offsetting compaction due to extensive supercoiling by mitoxantrone molecules. This conclusion was confirmed by independent experiments with PM2 covalently closed-circular DNA--both native, negatively supercoiled and relaxed--with calf thymus topoisomerase, using ethidium for comparison. Ethidium caused a 21.3 +/- 3.6% length increase in nicked, open-circular PM2-DNA, or 2100 molecules bound per 10,300 base pairs. Mitoxantrone caused a 16.6% length increase in nicked PM2-DNA equivalent to approximately 1700 drug molecules per circle. Electron microscopic measurements on relaxed PM2-DNA with progressively increasing proportions of mitoxantrone (from 1.4:1 to 14:1 drug molecules per base pair) revealed the onset of formation of lacelike networks of DNA circles linked together. This phenomenon, which is not produced by bisantrene, is attributed to inter-DNA links by the charged side arms of mitoxantrone and is in accord with previous reports that mitoxantrone causes severe compaction and distortion of chromatin. Electron microscopic examination of the interaction of six additional mitoxantrone derivatives, two of which produced lacelike DNA networks, revealed strict structural requirements for this phenomenon.
The chemical structure and conformation of the new antitumor antibiotic saframycin R have been determined by high field 1H and 13C NMR as well as FAB mass spectrometry. Unlike other members of the saframycin family, saframycin R contains a reduced quinone ring bearing a glycolic ester moiety. Saframycin R exhibits acid promoted equilibrium and reversible covalent binding to DNA templates and, in the presence of a reducing agent, oxygen dependent single strand scission of supercoiled DNA. The extent of DNA scission is enhanced by in situ porcine carboxyl esterase or base catalyzed cleavage of the glycolic ester function plausibly by the release of the more reactive reduced saframycin A. This suggests that saframycin R may be regarded as a less toxic pro-drug for the active forms of saframycins A or S.