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T L James

Publications and source records attributed to T L James.

At least 109 records · Page 6Linked to original sources
PubMed1990

Two-dimensional COSY and two-dimensional NOE spectroscopy of d(AC)4.d(GT)4: extraction of structural constraints.

Pure absorption phase, proton two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (DQF-COSY) spectra were recorded for d(AC)4.d(GT)4. A full proton resonance assignment was made, except for the 5' and 5" protons. A new semiautomatic method for improved quantitation of 2D NOE peak intensities was developed, and its limitations and usefulness were examined. With this new method, 2D NOE intensity sets at several mixing times were obtained. Simulations of the 1'2', 1'2", and 2'3' DQF-COSY cross-peaks were compared with experimental data, establishing an alternating sugar pucker for the alternating purine-pyrimidine sequence. Scalar coupling constants for the sugar ring protons, derived from the fitting of the simulated spectra, are reported. Complete relaxation matrix analysis of the 2D NOE spectrum verified this alternating structure for all NOE interactions between nonexchangeable protons. Both the DQF-COSY and the 2D NOE results qualitatively indicate that the structure of d(AC)4.d(GT)4 resembles wrinkled D-DNA in aqueous solution.

Computer Simulation↗
PubMed1990

Solution structure studies of d(AC)4.d(GT)4 via restrained molecular dynamics simulations with NMR constraints derived from two-dimensional NOE and double-quantum-filtered COSY experiments.

The structure of d(AC)4.d(GT)4 is investigated by constrained molecular dynamics simulations. The constraints include proton pair distances derived from 2D NOE intensities by using the iterative relaxation matrix analysis algorithm MARDIGRAS and sugar pucker phases and amplitudes derived from double-quantum-filtered COSY spectra. Molecular dynamics runs on simulated intensity and distance sets as well as the experimental data were carried out to determine the effects of starting structure, distance constraint derivation, energy functions, and experimental errors on the end result. It was found that structural details could not be elucidated within about 1.5-A overall atomic deviation. This limitation is due in part to the accuracy of the experimental data but, more importantly, is attributable to the quantity of experimental constraints available and to imperfections in the force field utilized in the molecular dynamics calculations. Within the limits of the method, some structural characteristics of d(AC)4.d(GT)4 could be elucidated.

Computer Simulation↗
PubMed1990

Deoxyribose conformation in [d(GTATATAC)]2: evaluation of sugar pucker by simulation of double-quantum-filtered COSY cross-peaks.

Exchangeable and nonexchangeable proton and phosphorus resonances (11.75 T) of [d(GTATATAC)]2 in aqueous solution were assigned by using proton two-dimensional nuclear Overhauser effect (2D NOE) spectra, homonuclear proton double-quantum-filtered COSY (2QF-COSY) spectra, proton spin-lattice relaxation time measurements, and 31P1H heteronuclear shift correlation spectra. Due to the large line widths, it was not possible to directly extract vicinal proton coupling constant values from any spectrum including ECOSY or 2QF-COSY. However, comparison of quantitative 2QF-COSY spectral simulations with experimental spectra enabled elucidation of coupling constants. The scope and limitations of this approach were explored by computation and by use of experimental data. It was found that proton line widths exhibit some variability from one residue to the next as well as from one proton to the next within a residue and the exact line width is critical to accurate evaluation of coupling constants. Experimental 2QF-COSY spectra were not consistent with a rigid deoxyribose conformation for any of the nucleotide residues. A classical two-state model, with rapid jumps between C2'-endo (pseudorotation angle P = 162 degrees) and C3'-endo (P = 9 degrees) conformations, was able to account for the spectral characteristics of terminal residue sugars: 60% C2'-endo and 40% C3'-endo. However, the 2QF-COSY cross-peaks from the -TATATA- core could be simulated only if the classical two-state model was altered such that the dominant conformer had a pseudorotation angle at 144 degrees instead of 162 degrees. In this case, the major conformer amounted to 80-85%. Alternatively, the spectral data were consistent with a three-state model in which C2'-endo and C3'-endo conformations had the largest and smallest populations, respectively, but a third conformer corresponding to C1'-exo (P = 126 degrees) was present, consistent with recent molecular dynamics calculations. This alternative yielded populations of 50% (P = 162 degrees), 35% (P = 126 degrees), and 15% (P = 9 degrees) for the -TATATA- sugars. The spectral results indicate little variation of sugar pucker between T and A. Small differences in cross-peak component intensities and characteristic spectral distortions, however, do suggest some unquantified variation. 31P1H heteronuclear chemical shift correlation spectra manifested alternating chemical shifts and coupling constants suggestive of phosphodiester backbone conformational differences between TA and AT junctions.

Base Sequence↗
PubMed1990

Cerebral metabolite dynamics during temporary complete ischemia in rats monitored by time-shared 1H and 31P NMR spectroscopy.

The changes in cerebral phosphorus metabolites, intracellular pH, and lactate during 30 min of complete global ischemia and 2 h of reperfusion were monitored by time-shared 1H and 31P in vivo NMR spectroscopy in rats. After the induction of ischemia, intracellular pH decreased from 7.14 +/- 0.01 to 6.32 +/- 0.10, and lactate concentration increased from 1.6 +/- 0.4 to 15.8 +/- 2.5 mumol/g; ATP and phosphocreatine were totally depleted, while inorganic phosphate increased 715 +/- 47%. Within 1 h after blood flow was restored, high-energy phosphates and lactate levels had recovered close to baseline levels. The changes in intracellular pH and lactate levels during ischemia and reperfusion correlated well.

Adenosine Triphosphate↗
PubMed1990

Correlations between in vivo 31P NMR spectroscopy measurements, tumor size, hypoxic fraction and cell survival after radiotherapy.

Phosphorus metabolite levels were measured non-invasively using 31P magnetic resonance spectroscopy (MRS) in SCCVII/SF tumors, subcutaneously transplanted into the legs of unanesthetized C3Hf/Sed mice. Shortly after MRS measurements, tumors were irradiated with a single dose of 20 Gy, and cell survival and radiobiologic hypoxic fraction were determined with an in vitro cloning assay. Significant correlations were found between tumor size and surviving fraction, hypoxic fraction, pH, and phosphorus metabolite ratios. With increase of tumor size, surviving fraction and hypoxic fraction both increased, the ratios of inorganic phosphate and phosphomonoesters to nucleoside triphosphates (Pi/NTP and PME/NTP, respectively) and inorganic phosphate to phosphocreatine (Pi/PCr) increased and pH decreased. However, considerable heterogeneity of MRS spectral parameters, even in tumors of similar size, precluded accurate prediction of hypoxic fraction and cell survival after radiotherapy.

Animals↗
PubMed1990

Stability of brain intracellular lactate and 31P-metabolite levels at reduced intracellular pH during prolonged hypercapnia in rats.

The tolerance of low intracellular pH (pHi) was examined in vivo in rats by imposing severe, prolonged respiratory acidosis. Rats were intubated and ventilated for 10 min with 20% CO2, for 75 min with 50% CO2, and for 10 min with 20% CO2. The maximum PaCO2 was 320 mm Hg. Cerebral intracellular lactate, pHi, and high-energy phosphate metabolites were monitored in vivo with 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, using a 4.7-T horizontal instrument. Within 6 min after the administration of 50% CO2, pHi fell by 0.57 +/- 0.03 unit, phosphocreatine decreased by approximately 20%, and Pi increased by approximately 100%. These values were stable throughout the remainder of the hypercapnic period. Cerebral intracellular lactate, visible with 1H NMR spectroscopy in the hyperoxic state, decreased during hypercapnia, suggesting either a favorable change in oxygen availability (decreased lactate production) or an increase in lactate clearance or both. All hypercapnic animals awakened and behaved normally after CO2 was discontinued. Histological examination of cortical and hippocampal areas, prepared using a hematoxylin and eosin stain, showed no areas of necrosis and no glial infiltrates. However, isolated, scattered, dark-staining, shrunken neurons were detected both in control animals (no exposure to hypercapnia) and in animals that had been hypercapnic. This subtle histological change could represent an artifact resulting from imperfect perfusion-fixation, or it could represent subtle neurologic injury during the hypercapnia protocol. In summary, extreme hypercapnia and low pHi (approximately 6.5) are well tolerated in rats for periods up to 75 min if adequate oxygenation is maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis, Respiratory↗
PubMed1990

Opiate-receptor antagonist improves metabolic recovery and limits neurochemical alterations associated with reperfusion after global brain ischemia in rats.

Opiate-receptor antagonists improve behavioral, electrophysiologic and/or histologic outcome in various experimental models of central nervous system ischemia. To address the potential mechanism(s) by which opiate-receptor antagonists may exert their protective actions in cerebral ischemia, metabolic and biochemical changes were measured in brain of rats pretreated with the opiate-receptor antagonist nalmefene or vehicle and subjected to 60 min of global ischemia followed by 2 hr of reperfusion. 31P and 1H magnetic resonance spectroscopy were used to follow the metabolic changes during ischemia and reperfusion, after which brain tissue was frozen in situ. Biochemical assays included free fatty acids, thromboxane B2, ascorbate, vitamin E and amino acids. Nalmefene-treated animals showed more rapid and complete recovery of cellular bioenergetic state (as indicated by the phosphocreatine to inorganic phosphate ratio), tissue acidosis and lactate levels during reperfusion than placebotreated controls. Ischemia/reperfusion caused significant increases of fatty free acids and thromboxane, associated with significant decreases of ascorbate and glutamate; nalmefene pretreatment limited each of these changes. The degree of metabolic improvement as reflected by recovery of high energy phosphates and reduction of lactic acidosis were highly correlated with changes in tissue levels of arachidonate and glutamate. Thus, the beneficial effects of opiate-receptor antagonists in cerebral ischemia may be due, in part, to an ability to enhance metabolic recovery with associated, reduction in phospholipid hydrolysis and excitotoxin release.

Adenosine Triphosphate↗
PubMed1989

Binding of actinomycin D to [d(ATCGAT)]2: NMR evidence of multiple complexes.

Actinomycin D (actD) binds to the oligonucleotide [d(ATCGAT)]2 with a hypochromatic and red-shifted visible absorbance band compared to free drug and a CD spectrum with double negative bands at 460 and 385 nm. These spectral features are similar to those of the actD-[d(ATGCAT)]2 complex, while actD-[d(AT)5]2 gives spectra similar to those of free drug. Upon dilution or raising the temperature, the spectral characteristics accompanying complex formation disappear in the actD-[(ATCGAT)]2 sample but remain in the actD-[d(ATGCAT)]2 complex under the same experimental conditions. These results suggest that (a) sequence-specific binding of actD occurs with [d(ATCGAT)]2 but not with [d(AT)5]2, (b) the binding is not as strong as with [d(ATGCAT)]2, and (c) actD binds [d(ATCGAT)]2 with the same mechanism as it binds [d(ATGCAT)]2, i.e., by intercalation. From NMR spectra of the actD-[d(ATCGAT)]2 complex, three types of signals can be detected below 20 degrees C, one major and two minor ones. At higher temperatures, exchange between the two minor ones becomes fast enough that only one type of minor signal was seen. Partial resonance assignments were made by using 2D nuclear Overhauser effect (NOE) and 2D homonuclear Hartmann-Hahn (HOHAHA) experiments. Proton chemical shift changes of the major complex are consistent with actD chromophore ring intercalation between hexamer base pairs. Data from NOE-detected dipolar interactions between actD and [d(ATCGAT)]2 protons were interpreted in terms of a major complex with the actD chromophore ring system intercalated at the CG position and minor complexes with the drug intercalated off center at the GA positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1989

Androgen sensitivity of rat prostate carcinoma studied by 31P NMR spectroscopy, 1H MR imaging, and 23Na MR imaging.

31P NMR spectroscopy, 1H magnetic resonance (MR) imaging, and 23Na MR imaging were used to study the biochemical difference between nine hormone-sensitive and six hormone-resistant rat prostate cancers and to follow bioenergetic and morphologic changes subsequent to androgen deprivation in the hormone-sensitive model. Neither 1H nor 23Na MR image characteristics were useful in distinguishing androgen-sensitive from androgen-resistant prostate cancer nor in identifying androgen deprivation. 31P NMR spectroscopy did detect bioenergetic differences between the hormone-sensitive and hormone-resistant tumors. Baseline spectra showed a significantly higher PCr/ATP ratio (mean 0.86 +/- 0.09 SEM) for hormone-sensitive tumors than for hormone-resistant tumors (mean 0.26 +/- 0.07 SEM). By 3 days after androgen deprivation (orchiectomy (castration], PCr/ATP ratios had decreased noticeably; by 1 week, the decrease was statistically significant and remained so for the rest of the study (3 weeks). It appears that 31P NMR spectroscopy is useful in detecting androgen sensitivity of prostatic carcinoma.

Androgens↗
PubMed1989

Transrectal probe for 1H MRI and 31P MR spectroscopy of the prostate gland.

A transrectal probe for 1H imaging and 31P MR spectroscopy of the prostate gland is described. The probe is covered with plastic and consists of an insectional portion containing an rf coil and a handle containing a tuning and matching circuit. Using this transrectal probe, 1H MR images and the first 31P MR spectra of the in situ canine prostate were obtained. Serial alterations in prostate size and changes in the ratio of phosphomonoesters to ATP ratio following orchiectomy were observed. Transrectal images of the prostate appear to be superior to conventional whole body coil images in the canine. For the first time a device to obtain high signal-to-noise 1H images and 31P spectra of the in situ prostate has been constructed. This will allow the assessment of transrectal imaging and spectroscopy as tools for medical studies of prostate pathophysiology.

Administration, Rectal↗
PubMed1989

Assessment of male infertility: correlation between results of semen analysis and phosphorus-31 magnetic resonance spectroscopy.

To evaluate the usefulness of phosphorus-31 magnetic resonance spectroscopy (31P-MRS) in assessing male infertility, we compared it with conventional semen analysis. Specimens were obtained from otherwise healthy patient groups as follows: group A, 7 fertile control subjects; group B, 12 azoospermic men after vasectomy; and group C, 11 patients presenting for infertility evaluation. Correlations between established semen analysis parameters and the 31P-MRS-derived ratio of glycerylphosphorylcholine to total phosphate (GPC/TP) were investigated. Group A controls had a mean GPC/TC ratio of 0.10 +/- 0.05, which was the same as that of group C. With the exception of significantly lowered motility and normal morphology in group C (p less than 0.001 and 0.05, respectively) semen analysis parameters in these two groups were similar. In contrast, the GPC/TP ratio in group B (0.05 +/- 0.04) was significantly different from the control (p less than 0.05), which appropriately reflected complete vasal occlusion. The results suggest that a significant portion of seminal GPC is derived from epididymal secretion and that 31P-MRS is useful for monitoring the GPC/TP levels when assessing epididymal function and male infertility.

Glycerylphosphorylcholine↗
PubMed1989

P-31 MR spectroscopy in assessing testicular torsion: rat model.

To develop phosphorus-31 magnetic resonance (MR) spectroscopy as an indicator of testicular viability, unilateral 720 degrees torsion of the spermatic cord was performed in 11 Copenhagen rats. In six of 11 rats, detorsion was done 1 hour later. The authors used special surface coils to obtain P-31 MR spectra (at 2 T) from both tests, then correlated MR findings with those from gross morphologic and histologic examination. In the normal testis, P-31 MR spectra had prominent phosphomonoester (PME) and adenosinetriphosphate (ATP) peaks. Testicular torsion dramatically reduced ATP to almost undetectable levels and significantly decreased the PME/Pi at 1 hour (1.18 +/- 0.22) in nine rats. In two rats, however, no spectral changes were present. Of the six rats in which detorsion was performed, three showed immediate regeneration of ATP and a normal PME/Pi (2.87 +/- 0.06) 3 hours later; testicles in the other three rats did not recover (PME/Pi = 0.72 +/- 0.01). Because gross morphologic observations and histologic findings prior to detorsion were unable to differentiate viable from nonviable tests, these preliminary data suggest P-31 MR spectroscopy may help clinicians diagnose testicular torsion and assess testicular viability.

Animals↗
PubMed1989

Effects of perfusion pressure on energy and work of isolated rat hearts.

A chemomechanical study of hypertrophied hearts of 6-month-old spontaneously hypertensive rats (SHR) and that of age-matched Wistar-Kyoto (WKY) rats was carried out, analyzing the response of the heart to steady-state changes in coronary perfusion pressure. The ratio of heart (dry)-to-body (wet) weight of WKY rats was 0.37 +/- 0.02 (10(-3] and for SHR was 0.58 +/- 0.03 (10(-3] (p less than 0.01). In the apex-ejecting, isolated, pyruvate-perfused working hearts of WKY rats and SHR, coronary flow was constant when coronary perfusion pressure was set between 140 and 190 cm H2O (range of autoregulation). Coronary flow was perfusion pressure dependent when the coronary perfusion pressure was set below 110 cm H2O for both WKY rats and SHR. Cardiac output, developed pressure, rate of pressure development (dP/dt), and oxygen consumption were constant in the range of autoregulation but decreased in the direction of coronary flow when coronary flow was reduced by a drop in perfusion pressure. Similarly, the phosphorylation potential, phosphocreatine, adenosine triphosphate, and cyclic adenosine monophosphate were constant in the range of autoregulation but decreased directionally with coronary perfusion pressure below 110 cm H2O for both SHR and WKY rats. There was a significantly lower phosphorylation potential in SHR as compared with WKY rats when coronary perfusion pressure was reduced to 80 cm H2O. In the region of autoregulation, coronary flow and oxygen consumption were significantly less in SHR, although developed pressure was significantly greater at both high and low workloads.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Deoxyribose ring conformation of [d(GGTATACC)]2: an analysis of vicinal proton-proton coupling constants from two-dimensional proton nuclear magnetic resonance.

Exchangeable and nonexchangeable protons of [d(GGTATACC)]2 in aqueous cacodylate solution were assigned from two-dimensional nuclear Overhausser effect (2D NOE) spectra. With phase-sensitive COSY and double quantum filtered COSY (DQF-COSY) experiments, the cross-peaks resulting from deoxyribose ring conformation sensitive proton-proton vicinal couplings, i.e., all 1'-2', 1'-2", 2'-3', and 3'-4' couplings and six from 2"-3' couplings, were observed. From the cross-peak fine structure, the 2',2" proton assignments can be confirmed; coupling constants J1'2' and J1'2" and sums of coupling constants involving H2' and H2" for all residues and H3' for C8 were obtained. The DISCO procedure [Kessler, H., Muller, A., & Oschkinat, H. (1985) Magn. Reson. Chem. 23, 844-852] was used to extract individual 1'-2' and 1'-2" coupling constants. The sum of coupling constants involving H1' or H3' was measured from the one-dimensional spectrum where signal overlap is not a problem. Analysis of the resulting coupling constants and sums of coupling constants, in the manner of Rinkel and Altona [Rinkel, L. J., & Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649], led to the following conclusion: C2'-endo deoxyribose ring conformation is predominant for every residue, but a significant amount of C3'-endo conformation may exist, ranging from 14% to 30%.

Carbohydrate Conformation↗
PubMed1988

Beneficial effects of verapamil during metabolic acidosis in isolated perfused rat hearts.

Metabolic acidosis was produced in two groups of isolated, glucose-perfused beating rat hearts. The first group (control) was untreated whereas the second group was pretreated for 48 h by the addition of verapamil (1.2 g/L) to the drinking water. Untreated hearts all developed asystole during a 30 min perfusion with an acidotic substrate (pH = 6.8) or during subsequent reequilibration with physiologic substrate (pH = 7.4). Prior to asystole, all untreated hearts showed evidence of severe mechanical and biochemical deterioration evaluated by 31 P NMR spectroscopy. In contrast, hearts of treated rats showed less mechanical and metabolic deterioration, and all recovered during reequilibration. The mechanism of protection of verapamil against the effects of metabolic acidosis is unclear but appears to be related to preserved mitochondrial function by the drug and not to a reduced demand for energy.

Acidosis↗