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Biomedical subjects

D L Turner

Publications and source records attributed to D L Turner.

At least 19 recordsLinked to original sources
PubMed2000

Generation of neurons by transient expression of neural bHLH proteins in mammalian cells.

Basic helix-loop-helix (bHLH) transcription factors are known to function during mammalian neurogenesis. Here we show that transient transfection of vectors expressing neuroD2, MASH1, ngn1 or related neural bHLH proteins, with their putative dimerization partner E12, can convert mouse P19 embryonal carcinoma cells into differentiated neurons. Transfected cells express numerous neuron-specific proteins, adopt a neuronal morphology and are electrically excitable. Thus, the expression of neural bHLH proteins is sufficient to confer a neuronal fate on uncommitted mammalian cells. Neuronal differentiation of transfected cells is preceded by elevated expression of the cyclin-dependent kinase inhibitor p27(Kip1) and cell cycle withdrawal. This demonstrates that the bHLH proteins can link neuronal differentiation to withdrawal from the cell cycle, possibly by activating the expression of p27(Kip1). The ability to generate mammalian neurons by transient expression of neural bHLH proteins should create new opportunities for studying neurogenesis and devising neural repair strategies.

Animals↗
PubMed1999

Solution structure of plantaricin C, a novel lantibiotic.

Plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin, is a lantibiotic. One dehydroalanine, one lanthionine and three beta-methyl-lanthionine residues were found in its 27 amino acid sequence. The plantaricin C structure has two parts: the first comprises the six NH2-terminal residues, four of which are lysines, which confer a strong positive charge to this stretch. The amino acids in positions 7 and 27 form the lanthionine bridge, giving a globular conformation to the rest of the molecule. The beta-methyl-lanthionine bridges are established between residues 12-15, 13-18 and 23-26. This central region has a charge distribution compatible with an amphipathic alpha-helix, through which plantaricin C would become inserted into the membrane matrix of sensitive organisms, provoking the opening of pores and leakage of the cytoplasmic content.

Amino Acid Sequence↗
PubMed1998

Functional and mechanistic studies of cytochrome c3 from Desulfovibrio gigas: thermodynamics of a "proton thruster".

Nuclear magnetic resonance and visible spectroscopies were used to determine the thermodynamic parameters of the four hemes in cytochrome c3 from Desulfovibrio gigas at 298 and 277 K and to investigate the mechanism of electron/proton energy transduction. Data obtained in the pH range from 5 to 9 were analyzed according to a model in which the hemes interact with each other (redox cooperativities) and with an ionizable center (redox-Bohr cooperativities). The results obtained at the two temperatures allow the deconvolution of the entropic contribution to the free energy of the four hemes, to the acid-base equilibrium of the ionizable center, and to the network of cooperativities among the five centers. The redox potentials of the hemes are modulated by the enthalpic contribution to the free energy, and evidence for the participation of the propionates of heme I in the redox-Bohr effect is presented. The network of interactions between the centers in this protein facilitates the concerted transfer of electrons and protons, in agreement with the "proton thruster" mechanism proposed for electronic to protonic energy transduction by cytochromes c3.

Cytochrome c Group↗
PubMed1998

Solution structure of Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3: structural basis for functional cooperativity.

Desulfovibrio vulgaris cytochrome c3 is a 14 kDa tetrahaem cytochrome that plays a central role in energy transduction. The three-dimensional structure of the ferrocytochrome at pH 8.5 was solved through two-dimensional 1H-NMR. The structures were calculated using a large amount of experimental information, which includes upper and lower distance limits as well as dihedral angle restraints. The analysis allows for fast-flipping aromatic residues and flexibility in the haem plane. The structure was determined using 2289 upper and 2390 lower distance limits, 63 restricted ranges for the phi torsion angle, 88 stereospecific assignments out of the 118 stereopairs with non-degenerate chemical shifts (74.6%), and 115 out of the 184 nuclear Overhauser effects to fast-flipping aromatic residues (62.5%), which were pseudo-stereospecifically assigned to one or the other side of the ring. The calculated NMR structures are very well defined, with an average root-mean-square deviation value relative to the mean coordinates of 0.35 A for the backbone atoms and 0.70 A for all heavy-atoms. Comparison of the NMR structures of the ferrocytochrome at pH 8.5 with the available X-ray structure of the ferricytochrome at pH 5.5 reveals that the general fold of the molecule is very similar, but that there are some distinct differences. Calculation of ring current shifts for the residues with significantly different conformations confirms that the NMR structures represent better its solution structure in the reduced form. Some of the localised differences, such as a reorientation of Thr24, are thought to be state-dependent changes that involve alterations in hydrogen bond networks. An important rearrangement in the vicinity of the propionate groups of haem I and involving the covalent linkage of haem II suggests that this is the critical region for the functional cooperativities of this protein.

Amino Acid Sequence↗
PubMed1998

Symmetry and phase-selected NMR spectra of liquid crystalline samples.

It is demonstrated that the NMR spectra of liquid crystalline samples can be simplified by using multiple quantum filtering. In a system of N spin-12 nuclei, the N or (N-1)-multiple quantum filtered spectra (NQF or (N-1)QF) contain lines which originate only from transitions among the eigenstates belonging to the highest symmetry class of the spin permutation group. In addition the NQF spectra are divided further into two sets of lines which differ in phase by 180 degrees. A method for simulating and analysing multiple quantum filtered spectra is described, with examples from molecules with up to eight interacting spins.

Magnetic Resonance Spectroscopy↗
PubMed1998

Determination of solution structures of paramagnetic proteins by NMR.

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron -nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8.

Anisotropy↗
PubMed1998

In vitro chemosensitivity of chronic lymphocytic leukaemia to purine analogues--correlation with clinical course.

Samples from 51 chronic lymphocytic leukaemia (CLL) patients (42 typical, nine atypical) were assessed for in vitro response to fludarabine and cladribine (2-CdA) using the flow cytometric terminal deoxynucleotidyl transferase (TdT) assay. No difference was demonstrated between the in vitro response of typical and atypical CLL and previous treatment did not result in a more apoptosis resistant phenotype. The assay could not distinguish those patients who required subsequent treatment from those whose disease remained stable, and universal cross-resistance/sensitivity to the two purine analogues was demonstrated. The assay's potential for use in the rapid assessment of in vivo response to purine analogue therapy in CLL was limited; correctly predicting the clinical outcome of 10/12 patients to treatment but failing to predict progression in two p53 deficient patients. The level of bcl-2 in the clonal lymphocytes did not influence the in vitro, spontaneous or drug-induced, apoptosis.

Antineoplastic Agents↗
PubMed1997

pH dependence of structural and functional properties of oxidized cytochrome c" from Methylophilus methylotrophus.

Cytochrome c" from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c" have been investigated over the pH range between 1.4 and 10.0. The rate of reduction displays proton-linked transitions of pKa congruent with 5.5 and 2.4, and a spectroscopic transition with a pKa congruent with 2.4 is also observed. The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry. Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (approximately 60%) of the alpha-helical content toward a random coil arrangement, which is recovered upon increasing the ionic strength. The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s). Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.

Circular Dichroism↗
PubMed1997

Paramagnetic NMR shifts in cyanoferricytochrome c. Investigation of thermal stability and deviations from Curie law behaviour.

The paramagnetic shifts of 13C nuclei positioned alpha to the haem in cyanoferricytochrome c are reported and analysed in terms of molecular orbitals based on D4h symmetry with a rhombic perturbation. The temperature dependence of the Fermi contact and dipolar shifts of the haem and axial histidine ligand show deviations from Curie Law behaviour which are explained by a Boltzmann distribution between partially filled 3e(pi) molecular orbitals and the ground and first excited state Kramers doublets. The comprehensive explanation of the temperature dependence of the paramagnetic shifts leads to the conclusion that there is no detectable temperature dependence of the haem orientation or that of the His ligand orientation. This work also provides evidence for the role of the axial His ligand in determining the orientation of the magnetic z-axis.

Cytochrome c Group↗
PubMed1997

Use of paramagnetic NMR probes for structural analysis in cytochrome c3 from Desulfovibrio vulgaris.

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) (c3DvH) is determined by means of a novel procedure. In this method the 13C chemical shifts of the nuclei directly bound to the haems are used to determine the in-plane orientations of the rhombic perturbation in each of the four haems with respect to a model of molecular orbitals of e(g) symmetry which are subject to a rhombic perturbation [Turner, D. L., Salgueiro, C. A., Schenkels, P., LeGall, J. & Xavier, A. V. (1995) Biochim. Biophys. Acta 1246, 24-281. These orientations, together with the components of the magnetic susceptibility tensors obtained from the EPR g values and the crystal structure of c3DvH, can be used to calculate the dipolar shifts induced by each haem throughout the protein. Thus the observed 13C paramagnetic shifts of the c3DvH haem substituents were fitted considering both the pseudocontact and contact shifts of each haem simultaneously. The dipolar shifts calculated by this method were tested against the observed dipolar shifts for some amino acid residues strategically placed in the protein and also for the haem propionate groups. The effect of considering the calculated dipolar extrinsic shifts on the behaviour of the chemical shifts of the haem methyl groups in the intermediate stages of oxidation at different pH values was also analysed. The several tests applied to the calculated dipolar shifts have shown that the method is extremely useful for predicting chemical shifts as an aid to complete proton assignment, and to add further constraints in the refinement of solution structures of paramagnetic proteins and hence to probe subtle structural rearrangements around the haem pocket.

Amino Acids↗
PubMed1997

Long-term facilitation of ventilation following repeated hypoxic episodes in awake goats.

1. This study tested two hypotheses: (1) that episodic hypoxia elicits long-term facilitation (LTF) in respiratory neurons that is manifest as an increase in ventilation in awake goats; and (2) that LTF causes complex changes in respiratory pattern which are responsible for the increase in ventilation. 2. Each goat participated in two protocols. In the first, inspired gas mixtures were alternated between isocapnic normoxia and hypoxia (arterial partial pressure of oxygen, Pa,O2 = 47 mmHg) for ten cycles. Each hypoxic episode lasted 3 min and normoxic intervals were 5 min. Ventilatory variables were measured during the last minute of each episode and periodically for up to 1 h following the last hypoxic episode. The second, sham protocol was undertaken at least 2 weeks later and was identical to the first, except that isocapnic hypoxia was replaced with normoxia. 3. Inspired ventilation (VI) increased during the first isocapnic hypoxic episode and reached progressively higher levels in subsequent hypoxic episodes. VI also increased progressively among normoxic intervals, such that by the tenth normoxic interval, it had increased 68% relative to the comparable sham value (P < 0.05). Respiratory frequency (FR), tidal volume and mean inspiratory flow all contributed to the augmented VI during both isocapnic normoxia and hypoxia. The increase in VI lasted up to 40 min after the final hypoxic episode, with an increased FR making the greatest contribution. The persistent increase in VI strongly suggests that episodic hypoxia elicits LTF in respiratory neurons in the awake goat. Complex changes in respiratory pattern underpin the ventilatory manifestation of LTF.

Animals↗
PubMed1997

Assignment of the ligand geometry and redox potentials of the trihaem ferricytochrome c3 from Desulfuromonas acetoxidans.

Cytochrome c551.5 is a trihaem cytochrome of the cytochrome c3 family isolated from Desulfuromonas acetoxidans. Although several X-ray structures are available for tetrahaem cytochromes of this family, there is no X-ray structure for trihaem cytochromes. Cytochrome C551.5 was studied in the oxidized form by means of two-dimensional NMR. The pattern of observed interhaem NOESY connectivities is in agreement with the haem core structure previously determined by NMR for the reduced protein [Coutinho, I. B., Turner, D. L., Liu, M. Y., LeGall, J. & Xavier, A. V. (1996) J. Biol. Inorg. Chem. 1, 305-311]. The similarities found between the haem core structure and the amino acid sequence of cytochrome c551.5 and those of tetrahaem cytochromes c3 allows each of the haems to be specifically assigned in the polypeptide sequence, and the attribution of the midpoint redox potentials to the individual haems. This also allows individual redox potentials to be assigned to each haem in the NMR spectrum. The paramagnetic shifts of the 13C resonances of the haem substituents were analyzed in terms of pi molecular orbitals with perturbed D4h symmetry. The parameters of this analysis have been shown to be controlled by the orientation of the axial ligands in several other bis-His-coordinated haems and hence the ligand geometry was deduced for cytochrome C551.5. The structural analogy between the relative haem plane orientations in cytochrome c551.5 and the tetrahaem cytochromes c3 is found to extend to the axial ligands with the largest differences being in the vicinity of the deleted fourth haem, using the numbering of cytochrome c3 haems.

Bacteria↗
PubMed1997

Modulation of ventilatory control during exercise.

The control of ventilatory responses to mild or moderate dynamic exercise has been the subject of considerable debate for over a century. The prevailing view has been that the ventilatory response to exercise is stereotypical and rather unmalleable. However, paradigms involving novel associations of stimulus inputs have been shown to modulate breathing in short and longer time scales. The scope of this review includes examples of modified ventilatory responses to exercise which have been investigated in terms of neural mechanisms. An attempt to synthesise the available data into a model of neuromodulation is presented.

Animals↗
PubMed1997

Effects of endurance training on oxidative capacity and structural composition of human arm and leg muscles.

Six healthy subjects performed endurance training of the same duration with legs and arms consecutively. Performance and muscle structure were measured before and after training in lower and upper limbs. Training induced similar increases in maximal oxygen consumption (6 +/- 1 vs. 7 +/- 2 mL min-1 kg-1: legs vs. arms, P > 0.05) and mitochondrial volume in leg and arm muscles (42 +/- 12 vs. 31 +/- 11%: legs vs. arms, P > 0.05). The gain in mitochondrial volume after training was achieved solely by increasing the fraction of mitochondria (+40 +/- 11%, P < 0.05) in the same muscle volume (+2 +/- 2%, P > 0.05) in the legs. In contrast, increased muscle volume (+14 +/- 3%, P < 0.05), in addition to a tendency for an increase in mitochondrial fraction (+16 +/- 11%, P > 0.05), occurred in the arms after training. Thus, similar improvements in muscle oxidative capacity in upper and lower limbs were brought about by different mechanisms. It is suggested that due to infrequent use and a lack of load-bearing function, arm muscle volume is underdeveloped in untrained, sedentary or detrained/injured subjects and that the mode of endurance training used in this study is sufficient to enlarge arm muscle volume as well as aerobic capacity.

Adult↗
PubMed1997

The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis.

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.

Animals↗
PubMed1996

NMR studies of cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris.

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc3) are rationalised by a model which involves both homotropic (e-/e-) and heterotropic (e-/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the Dvc3 have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.

Allosteric Regulation↗