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A Tucker

Publications and source records attributed to A Tucker.

At least 55 records · Page 3Linked to original sources
PubMed1997

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin.

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3' position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5'-adenylyl-beta, gamma-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.

Binding Sites↗
PubMed1996

Solution structure of a parallel left-handed double-helical gramicidin-A determined by 2D 1H NMR.

The structure of a parallel left-handed double-helical form of gramicidin was detected by circular dichroism spectroscopy and determined using 500 and 600 MHz NMR in CaCl2/methanol solution. Measurements of TOCSY, DQF-COSY and NOESY spectra were converted into 604 distance and 48 torsional angle constraints for structure calculations. Stereospecific assignments and chi 1 angles were calculated using 3J alpha beta, d alpha beta (i,i), dN beta(i,i) and dN gamma(i,i). chi 2 angles were determined using d alpha beta(i,i), dN beta(i,i), d beta delta(i,i), dN gamma(i,i) and d alpha gamma(i,i). The calculations of initial structures were performed using the distance geometry/simulated annealing method in XPLOR. The initial structures were further refined and energy minimized using simulated annealing/molecular dynamics methods. Back-calculations for every generated structure were also performed to check their consistency with the experimental data. 187 final structures with no violations above the threshold conditions (0.05 A, 5 degrees, 5 degrees, 0.5 A and 5 degrees for bonds, angles, improper, NOE and cdihe, respectively) were produced from the 200 initial structures. Twenty structures with the lowest NOE energies were used for further analysis. The average r.m.s. deviations for the 20 structures are 0.64 A for backbone and 1.1 A for all non-hydrogen atoms. Gramicidin in this form, with approximately 5.7 residues per turn, is a parallel double helical dimer. The length along the helix axis is about 30 A and the inner pore diameter varies from 1 to 2 A. It is different from all other gramicidin structures determined to date. The presence of Ca2+ stabilises a conformation that prevents the binding of monovalent cations. It is likely that this structure is related to a non-channel, antibiotic role of gramicidin.

Amino Acid Sequence↗
PubMed1996

Cranial motor axons respond differently to the floor plate and sensory ganglia in collagen gel co-cultures.

Within the developing chick hindbrain, motor neurons differentiate in columns on either side of the ventral midline floor plate. Along the rostrocaudal axis, populations of motor neurons are organized segmentally with the trigeminal (V) and facial (VII) nuclei occupying successive pairs of rhombomeres. To reach their targets, motor axons follow stereotyped pathways. Branchiomotor and visceral motor axons of the Vth and VIIth nerves first project in a dorsal (lateral) direction away from the floor plate and towards the nerve exit point located in the alar plate of the even-numbered rhombomere of the pair. Having exited the hindbrain, axons grow in association with the cranial sensory ganglia before branchiomotor axons enter the branchial arches. We have investigated some of the factors that might guide cranial motor axons using a three-dimensional collagen gel culture system. When explants of hindbrain basal plate containing trigeminal or facial motor neurons were co-cultured with floor plate explants, axon outgrowth from the side facing the floor plate was inhibited in a manner consistent with chemorepulsion. When basal plate explants that contained an exit point were cultured alone, motor axons grew to the exit point and then stopped. When basal plate explants were co-cultured with trigeminal ganglia, motor outgrowth was increased in comparison with that in control cultures, suggesting a trophic influence. The findings presented here indicate that motor pathways are elaborated due to a progression of signals to which the growth cones respond in sequence.

Animals↗
PubMed1996

Endotoxin-induced adhesion of red blood cells to pulmonary artery endothelial cells.

Cell-cell interactions are important in intravascular inflammation. Neutrophils and monocytes adhere to the vascular endothelium and release mediators, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, and reactive oxygen species. Red blood cells (RBC) from patients with malaria, sickle cell anemia, and diabetes also adhere to endothelial cells. The objectives of this investigation were to develop a bovine system of RBC adhesion to endothelial cells and to begin to investigate the mechanisms involved in the RBC adhesion. We show that 51Cr-RBC adhere to bovine pulmonary artery endothelial cells (BPAEC) after stimulation of both cell types with endotoxin (ETX; 50 micrograms/ml). RBC adhesion to BPAEC depended on the ETX concentration and the presence of divalent cations. TNF-alpha, IL-1 beta, and antioxidants (superoxide dismutase; catalase; and dimethyl sulfoxide) all induced RBC adhesion to BPAEC. Phosphatidylserine, which has been implicated in adhesion of sickle cells and aged RBC to endothelium, reduced RBC adhesion to BPAEC, whether ETX-treated or not. In conclusion, ETX, proinflammatory cytokines and, surprisingly, antioxidants increase RBC adherence to BPAEC monolayers. RBC adhesion to endothelium is decreased by phosphatidylserine.

Animals↗
PubMed1996

Women have lower tolerance to lower body negative pressure than men.

Studies of the cardiovascular response to lower body negative pressure (LBNP) in men and women have suggested that women may have less tolerance to LBNP than men, although tolerance per se was not determined. To investigate the effect of gender on tolerance to LBNP, 10 men 10 women were subjected to increasing levels of LBNP until presyncopal symptoms developed. The cumulative stress index (CSI) score was determined, as were cardiovascular variables. Women had 62% less tolerance to LBNP with a CSI of 412 +/- 43 mmHg/min compared with a CSI of 1,070 +/- 149 mmHg/min for men. Cardiovascular changes associated with LBNP were similar for men and women when expressed relative to the occurrence of presyncope, but women had a higher heart rate response when the data were expressed at absolute levels of LBNP (-30 and -50 mmHg LBNP). Thus men and women had similar cardiovascular adjustments to the LBNP, with the changes in women occurring lower levels of LBNP. These data are important in a consideration of the development of antigravitational countermeasures for women. These data raise questions as to the manner in which blood pools within the lower body in men and women under LBNP.

Adult↗
PubMed1995

1H NMR of albumin in human blood plasma: drug binding and redox reactions at Cys34.

1H NMR methods are described which allow direct studies of the Cys34 binding site of albumin in intact human blood plasma in vitro. Antiarthritic gold drugs and the alcohol-aversive drug disulfiram induce a structural transition detectable via H epsilon 1 and H delta 2 resonances of His3 of albumin, and reactions of cystine, glutathione and captopril in plasma have also been investigated. Contrary to most assumptions, little of the albumin in normal plasma appears to be blocked at Cys34 as a cystine disulfide.

Alcohol Deterrents↗
PubMed1995

Site-specific N-terminal auto-degradation of human serum albumin.

Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30 degree C. Mass spectrometry and N-terminal sequencing of the protein identified degradation corresponding to the loss of the first two residues, aspartic acid and alanine. The reaction was shown to be dependent upon temperature and the N-terminal alpha-amino group. In addition, comparison with serum albumins derived from other species showed that the instability of the N-terminus was specific to the human albumin sequence. An intact aspartyl-alanyl dipeptide, purified from degraded albumin solutions, differed substantially from a synthetic dipeptide on amino acid analysis, N-terminal sequencing and NMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.

Amino Acid Sequence↗
PubMed1995

Spontaneous preoperative internal jugular and subclavian vein thrombosis associated with an early-stage synchronous ovarian/endometrial malignancy.

A case of preoperative spontaneous internal jugular/subclavian vein thrombosis documented with magnetic resonance imaging associated with a synchronous stage II ovarian/stage I endometrial malignancy is presented. This unusual deep venous thrombosis site is classically associated with trauma, infection, head and neck malignancies, or central venous catheterization and is rarely associated with distant malignancies. Neck pain and swelling in a gynecologic oncology patient should prompt consideration of this diagnosis.

Carcinoma, Endometrioid↗
PubMed1995

Static tension is associated with increased smooth muscle cell DNA synthesis in rat pulmonary arteries.

During the development of pulmonary hypertension, vascular cell proliferation closely parallels the rise in pulmonary intravascular pressure. The possible direct physical effect that elevated pressures may have on inducing vascular cell proliferation in pulmonary hypertension is unclear. To address this question, static force (0, 1, 5, and 10 g) was applied to hilar pulmonary arterial rings cultured in a serum-free medium. Incorporation of the thymidine analogue, bromodeoxyuridine (BrdU), into medial and adventitial cells was analyzed by immunohistochemistry. Medial cell BrdU incorporation (%positive cells) was increased (P < 0.0001) at all levels of force compared with 0-g controls (unmounted and mounted, but without applied force) (unmounted: 0.65 +/- 0.08; mounted: 0 g, 1.8 +/- 0.39; 1 g, 3.7 +/- 0.35; 5 g, 5.2 +/- 0.43; 10 g, 2.8 +/- 0.17). Hypoxia exposure and endothelial denudation of arteries attenuated (P < 0.05) tension-induced medial cell BrdU labeling (2.5 +/- 0.96 and 3.3 +/- 0.63, respectively) compared with control arteries (6.0 +/- 0.54). Nifedipine reduced tension-induced medial cell BrdU incorporation (P < 0.05). There was no difference in DNA synthesis in adventitial cells at the various levels of force, although hypoxia decreased adventitial cell BrdU incorporation overall (P < 0.05). We conclude that static wall tension may be an important direct stimulus for medial cell DNA synthesis.

Animals↗
PubMed1995

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Education, Nursing, Continuing↗
PubMed1994

A new structural transition of serum albumin dependent on the state of Cys34. Detection by 1H-NMR spectroscopy.

1. Reactions of fatty-acid-free bovine serum albumin and recombinant human albumin with a range of antiarthritic gold(I) complexes [auranofin, deacetylated auranofin, triethylphosphinegold(I) chloride] and related thiols (thioglucose, tetraacetylthioglucose, glutathione, dithiothreitol) have been investigated using 1H-NMR spectroscopy. 2. In reactions of albumin with auranofin, tetraacetylthioglucose and dithiothreitol, release of cystine was detected, whereas for deacetylated auranofin, thioglucose and glutathione, mixed disulphides with cysteine were produced. It has been previously proposed that Cys34 of human and bovine serum albumins is partly blocked by disulphide formation with cysteine and glutathione. The above reactions lead to deblocking by thiol-disulphide interchange reactions. No release of glutathione from albumin was detected. 3. Changes in the His H epsilon 1 regions of the 1H-NMR spectra show that albumin exists in two structural forms dependent on whether the side-chain of Cys34 is a free thiolate, or blocked by gold(I)triethylphosphine, by disulphide formation with cysteine or by another form of oxidation. We propose that Cys34 is either in a buried or in an exposed environment; the possible molecular basis of the structural change is discussed. 4. The relationship between reactions at Cys34, cysteine release, and the observed structural transition are discussed in terms of chrysotherapy, albumin metabolism and the use of gold(I) as a heavy atom derivative in X-ray crystallographic studies of albumins.

Amino Acid Sequence↗
PubMed1994

Adenosinergic modulation of CA1 neuronal tolerance to glucose deprivation in organotypic hippocampal cultures.

Glucose deprivation produced neuronal degeneration of CA1 pyramidal neurons in hippocampal slice cultures. The effects of the adenosine agonist cyclohexyladenosine (CHA) and antagonist cyclopentylxanthine (CPX) on CA1 neuronal loss following hypoglycemia was examined using propidium iodide fluorescence as an indicator of cell death. The intensity of propidium iodide fluorescence in hippocampal area CA1 was quantified using Optimas image analysis software. Following 2 or 3 h of glucose deprivation, CPX significantly enhanced injury in the CA1 region while CHA provided significant protection. These results suggest that adenosine plays an important role in endogenous neuronal protection during hypoglycemic injury, and also supports a role for the use of adenosine agonists as neuroprotective agents.

Adenosine↗
PubMed1994

pH-induced structural changes in human serum apotransferrin. pKa values of histidine residues and N-terminal amino group determined by 1H-NMR spectroscopy.

The binding of apotransferrin (80 kDa) to the transferrin receptor is known to be highly pH-dependent. We have investigated pH-induced structural changes in human serum apotransferrin over the pH* (meter reading in D2O solutions) range 2.5-11 using 1H-NMR spectroscopy. The pKa values of 14 (possibly 15) of the 19 His residues in the protein have been determined as well as that of the terminal amino group (Val1, 7.75). About eight His residues deprotonate when the pH* is raised from the endosomal value of about 5.5 to the blood plasma value (7.4). Four His residues have pKa < 6. Sharp discontinuities in the His titration curves were observed below pH 4.3 and at pH 3.5 molten globule states were detected.

Apoproteins↗
PubMed1994

Involvement of a lysine residue in the N-terminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+.

We report one-dimensional and two-dimensional 1H-NMR studies of the binding of Ni2+, Cu2+, Co2+, Cd2+ and Al3+ to defatted bovine and human serum albumins. The diamagnetic shifts induced by Ni2+, and paramagnetic effects due to Cu2+, were consistent with strong binding to a square-planar site formed by the three N-terminal amino acid residues (Asp-Thr-His for bovine, and Asp-Ala-His for human albumin). In contrast to previous studies on isolated 1-24 N-terminal peptide, a Lys residue also appeared to be involved in the binding site, and is assigned as Lys4. A second His residue is also close to the Cu2+/Ni2+ binding site in bovine serum albumin and is assigned to His59 (not present in human albumin). Co2+ caused specific perturbation of the resonances for the three N-terminal residues as well as those for Lys4. This is the first evidence for Co2+ binding to the N-terminal metal site of serum albumin. Neither Al3+ nor Cd2+ perturbed resonances for the N-terminal amino acids, but bind elsewhere in the protein.

Aluminum↗
PubMed1994

Residual lung volume and ventilatory muscle strength changes following maximal and submaximal exercise.

In order to evaluate a mechanism which may be responsible for the often observed acute increase in residual lung volume (RV) following exercise, 12 non-smoking males (20-30 yrs) performed two bouts of exercise on separate days; one to maximal heart rate (HRmax) and one to 85% of HRmax for 20 min. Prior to exercise and at 5, 15, 30, 60 and 120 min post-exercise, the following parameters were measured: RV, forced vital capacity (FVC), forced expiratory volume (FEV1.0), forced expiratory flow (FEF75-85), maximal expiratory pressure (PEmax), and maximal inspiratory pressure (PImax). Significant (p < 0.05) increases occurred in RV at 5, 15 and 30 min following maximal exercise and at 5 and 30 min after submaximal exercise. Changes in RV between the two exercise bouts were generally greater (p < 0.05) for maximal exercise. Accompanying the increases in RV were significant (p < 0.05) decreases in PEmax and decreases in FVC, while FEV1.0 and FEF75-85 remained generally unchanged or were slightly elevated. The data suggest that decreases in expiratory muscle strength due to fatigue may in part be responsible for increases in RV.

Adult↗