Biomedical subjects

D L Turner

Publications and source records attributed to D L Turner.

At least 19 recordsLinked to original sources

PubMed2003

The amplitude of the slow component of oxygen uptake is related to muscle contractile properties.

During constant-load exercise above the lactate threshold, oxygen-uptake kinetics deviate from the pattern seen below the threshold, with an additional, delayed component superimposed on the monoexponential pattern. It was hypothesised that this slow component is due to the progressive recruitment of type II muscle fibres. Oxygen uptake was measured for six male power athletes (group P) and six male endurance athletes (group E) during constant-load knee extension exercise tests in order to determine slow component amplitude. In addition, an electrical stimulation protocol was employed in order to assess the functional contractile profile and fatiguability of the knee extensors. The amplitude of the slow component during exercise was significantly ( P<0.05) greater in group P than in group E when expressed as an absolute value [mean (SEM)=77 (17) ml min(-1) and 24 (16) ml min(-1)] and when normalised to end-exercise oxygen uptake, VO(2) [8.2 (0.5)% and 2.6 (1.8)%]. Group differences were observed for percentage force loss during the electrical stimulation protocol [50.0 (3.4)% and 31.5 (3.7)% for groups P and E, respectively], increase in relaxation time from start to end of the fatigue test [87.9 (15.5)% and 31.1 (11.9)%], and relaxation time for fresh muscle [32.4 (1.0) ms and 40.6 (2.1) ms]. These contractile parameters may indicate a higher proportion of type II fibres in group P compared with group E. These experiments have shown evidence of a relationship between the amplitude of the slow component and muscle contractile properties, indicating that the origin of the slow component may lie in the pattern of different muscle fibre types.

Adult↗

PubMed2003

Evidence for an extensive collagen type III proximal domain in the rat femur. II. Expansion with exercise.

Exercise in youth may affect bone "quality" as well as quantity. Using the rat model, 1.5-month-old females were divided into four weight-matched groups, exercised short-term (6 weeks, E(s), n = 20) and long-term (14 weeks, E(L), n = 10) by access to monitored running wheels, and corresponding "sedentary" controls (S(S) short-term, n = 20; S(L) long-term, n = 10). Femora were either plastic-embedded or fresh-frozen. Transverse histological slices, 100 microm thick, were cut midshaft, while similar cryosections, 8 microm thick, were prepared from the same site and also coronal to the femoral neck region. An image analyser measured femoral neck and midshaft microarchitecture, while immunostaining localized collagen type III-rich fibres (CIII, an index of Sharpey fibre insertions) and osteopontin-rich osteons (OPN, an index of remodelling). Exercise increased cortical bone (proximal width +18%, midshaft area +7%). It also raised cancellous bone volume (+25%) by trabecular thickening (+30%) with more intraosseous vascularity and new trabecular interconnections (node-terminus ratio, +57%; trabecular pattern factor, -147%; marrow star volume. -48%). In the cortex a prominent discrete subperiosteal domain became wider (+50% midshaft) with exercise and contained more numerous (+15%) CIII-stained fibres. In contrast the encircled inner bone developed more numerous (+14%) OPN-rich osteons. It is concluded that short-term voluntary exercise augments both cortical and cancellous microarchitecture. It also alters protein composition, such that expanding arrays of Sharpey's fibres within a circumferential proximal domain (Part I) interconnect more powerfully with the musculature and interface more robustly with the core bone that in response becomes more vascular and biodynamic, providing further insight into how muscle mass may be skeletally translated.

Animals↗

PubMed2002

Different regional effects of voluntary exercise on the mechanical and electrical properties of rat ventricular myocytes.

Short-term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole-heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross-sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub-endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+](i) transients, or in myofilament Ca2+ sensitivity (assessed during re-lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross-sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension-sarcomere length relationship, suggesting an exercise-induced modulation of the Frank-Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub-epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T-wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.

Actin Cytoskeleton↗

PubMed2002

Predicting toxicity of tall larkspur (Delphinium barbeyi): measurement of the variation in alkaloid concentration among plants and among years.

Tall larkspur (Delphinium barbeyi) is the principal mountain larkspur responsible for the majority of cattle deaths on mountain rangelands in western Colorado and central and southern Utah in the United States. Ten plants in each of two tall larkspur populations in the mountains near Ferron and Salina, Utah, were marked, and single stalks were harvested periodically through the growing season for 4 yr. Toxic alkaloid concentration [alkaloids containing the N-(methylsuccimimido)-anthranilik ester group] was determined by Fourier transform infrared (FTIR) spectroscopy. Individual larkspur plants varied in alkaloid concentrations, especially in early growth (14-38 mg/g). As the concentration declined over the growing season, variation among plants also declined. There were yearly differences in alkaloid concentration among individual plants (P < 0.01) and populations (P < 0.001), even after accounting for differences in phenological growth between years. Variables such as precipitation, temperature, days since snow melt, growing degree days (sum of mean temperature each day from snow melt), and plant height and weight were all considered in a Mallows Cp multiple regression selection procedure to predict alkaloid concentration. The mixed model procedure in SAS adjusted the regression equation for locations and years. Growing degree days was the best single predictor of alkaloid levels: In y = (3.581 - 0.00423 GDD), R2 = 0.85. Internal validation of this equation within individual years and locations from which the equation was developed, produced correlations between observed versus predicted values ranging from r = 0.73 to 0.93. External validations on nine other larkspur populations produced correlations ranging from r = 0.76 to 0.99. This predictive equation can provide a tool for ranchers and land managers to make management decisions of when to graze cattle in larkspur areas.

Alkaloids↗

PubMed2001

Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning.

The basic-helix-loop-helix (bHLH) proteins are a superfamily of DNA-binding transcription factors that regulate numerous biological processes in both invertebrates and vertebrates. One family of bHLH transcriptional repressors is related to the Drosophila hairy and Enhancer-of-split proteins. These repressors contain a tandem arrangement of the bHLH domain and an adjacent sequence known as the Orange domain, so we refer to these proteins as bHLH-Orange or bHLH-O proteins. Phylogenetic analysis reveals the existence of four bHLH-O subfamilies, with distinct, evolutionarily conserved features. A principal function of bHLH-O proteins is to bind to specific DNA sequences and recruit transcriptional corepressors to inhibit target gene expression. However, it is likely that bHLH-O proteins repress transcription by additional mechanisms as well. Many vertebrate bHLH-O proteins are effectors of the Notch signaling pathway, and bHLH-O proteins are involved in regulating neurogenesis, vasculogenesis, mesoderm segmentation, myogenesis, and T lymphocyte development. In this review, we discuss mechanisms of action and biological roles for the vertebrate bHLH-O proteins, as well as some of the unresolved questions about the functions and regulation of these proteins during development and in human disease.

Amino Acid Sequence↗

PubMed2001

Cooperativity between electrons and protons in a monomeric cytochrome c(3): the importance of mechano-chemical coupling for energy transduction.

To fully understand the structural bases for the mechanisms of biological energy transduction, it is essential to determine the microscopic thermodynamic parameters which describe the properties of each centre involved in the reactions, as well as its interactions with the others. These interactions between centres can then be interpreted in the light of structural features of the proteins. Redox titrations of cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 followed by NMR and visible spectroscopy were analysed by using an equilibrium thermodynamic model. The network of homotropic and heterotropic cooperativities results in the coupled transfer of electrons and protons under physiological conditions. The microscopic characterisation allows the identification of several pairs of centres for which there are clear conformational (non-Coulombic) contributions to their coupling energies, thus establishing the existence of localised redox- and acid-base-linked structural modifications in the protein (mechano-chemical coupling). The modulation of interactions between centres observed for this cytochrome may be an important general phenomenon and is discussed in the framework of its physiological function and of the current focus of energy transduction research.

Acid-Base Equilibrium↗

PubMed2001

Conformational component in the coupled transfer of multiple electrons and protons in a monomeric tetraheme cytochrome.

Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c(3) is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechano-chemical coupling involved in the transfer of multiple electrons and protons in cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.

Cytochrome c Group↗

PubMed2001

Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.

Cytochrome c Group↗

PubMed2001

Thermodynamic control of electron transfer rates in multicentre redox proteins.

In the analysis of kinetic data from multicentre redox proteins, it is essential to distinguish between the observable macroscopic rate constants and the structurally relevant microscopic properties. This distinction is complicated by the existence of interactions between centres. The problem is illustrated by the case of two interacting redox centres and generalised for the analysis of stopped-flow kinetic data for the reduction of cytochrome c(3), in which four redox centres and at least one proteolytic centre are mutually interacting. It is shown that fast intramolecular electron transfer, which is typical of many multicentre redox proteins, and, where present, fast proton exchange, ensure that only N rate constants can be measured for a protein with N redox centres. The equations that relate the observable macroscopic rate constants to the microscopic rate constants of individual centres depend on a set of parameters that can be approximated by using the Marcus theory of electron transfer together with a set of reasonable assumptions. The results are tested by fitting experimental data for the reduction of cytochrome c(3) by sodium dithionite, including its pH dependence.

Binding Sites↗

PubMed2001

Solution structure of Methylophilus methylotrophus cytochrome c": insights into the structural basis of haem-ligand detachment.

Cytochrome c" from Methylophilus methylotrophus is a monohaem protein with 124 amino acid residues. The iron has two histidine ligands in the oxidised form, one of which detaches and picks up a proton when the protein is reduced. Thus, both forms are paramagnetic. The structure of the oxidised form in solution, determined from NMR data is presented. The family of structures has an average backbone rmsd value of 0.53 A, and a heavy atom rmsd value of 0.95 A, within a target function range of 32 %. This structure is related to class I cytochromes with an additional helix at the N terminus. The haem-binding site occurs in a domain essentially lacking secondary structure motifs and the axial histidinyl residues were found in an unusual near perpendicular orientation. Moreover, a disulfide bridge is present, an uncommon structural feature among c-type cytochromes. The disulfide bridge, linking cysteine residues 96 and 104, forms a loop that confers rigidity and is essential to the detachment of the axial histidine (His95) as demonstrated by chemical disruption of the S-S bond. A route for protonation of the distal histidine involving haem propionate 17 is proposed and discussed in the light of available models for complex membrane proton pumps.

Amino Acid Sequence↗

PubMed2001

NMR structure of the haem core of a novel tetrahaem cytochrome isolated from Shewanella frigidimarina: identification of the haem-specific axial ligands and order of oxidation.

The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c(3) so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c(3) isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.

Amino Acid Sequence↗

PubMed2001

Notch signaling can inhibit Xath5 function in the neural plate and developing retina.

Neuronal differentiation is regulated by both positive and negative regulatory factors; however, precisely how these factors interact to regulate retinogenesis is still unclear. We have examined the ability of the Notch pathway to modulate the function of the basic helix-loop-helix factor Xath5. Overexpression of Xath5 by RNA injection into cleavage-stage blastomeres promotes ectopic neurogenesis at neural plate stages and ganglion cell differentiation in the developing retina. We found that these activities of Xath5 could be inhibited by coexpression of activated Notch. Notch inhibition of Xath5 function was reversed by coexpression with the zinc finger protein X-MyT1. The Notch effector enhancer-of-split related 1 (ESR1) also blocked Xath5 activity but efficient inhibition by ESR1 required the DNA binding basic domain and the conserved WRPW motif. In addition, ESR1 inhibited the ability of Xath5 to directly activate the expression of XBrn3d, a transcription factor involved in retinal ganglion cell development. Xath5 could upregulate expression of X-Delta-1, ESR1, and ESR3, suggesting that Xath5 participates in a regulatory loop with the Notch pathway.

Amino Acid Motifs↗

PubMed2001

NMR structure of Desulfovibrio gigas rubredoxin: a model for studying protein stabilization by compatible solutes.

Rubredoxins are small, soluble proteins that display a wide variation in thermostability, despite having a high degree of sequence similarity They also vary in the extent to which they are stabilized by solutes such as diglycerol phosphate. Hence, they provide excellent models for studying the mechanisms of thermostabilization. Nuclear magnetic resonance (NMR) spectroscopy can be used to investigate interactions between molecules, as well as subtle changes in conformation in solution, and also provides a means to measure protein stability. The assignment of the proton NMR spectrum of the zinc rubredoxin from Desulfovibrio gigas is presented, together with its structure in solution. The stabilizing effect of diglycerol phosphate on rubredoxin is demonstrated and assessed by determining selected amide proton exchange rates; diglycerol phosphate at 100 mM concentration caused an additional structural stabilization of 1.2 +/-0.4 kJ/mol. The pattern of effects on the exchange rates is discussed in relation to the protein structure.

Amides↗

PubMed2001

Molecular targets of vertebrate segmentation: two mechanisms control segmental expression of Xenopus hairy2 during somite formation.

Vertebrate hairy genes are expressed in patterns thought to be readouts of a "segmentation clock" in the presomitic mesoderm. Here we use transgenic Xenopus embryos to show that two types of regulatory elements are required to reconstitute the segmental pattern of Xenopus hairy2. The first is a promoter element containing two binding sites for Xenopus Su(H), a transcriptional activator of Notch target genes. The second is a short sequence in the hairy2 3' untranslated region (UTR), which most likely functions posttranscriptionally to modulate hairy2 RNA levels. 3' UTRs of other hairy-related, segmentally expressed genes can substitute for that of hairy2. Our results demonstrate a novel mechanism regulating the segmental patterns of Notch target genes and suggest that vertebrate segmentation requires the intersection of two regulatory pathways.

3' Untranslated Regions↗

PubMed2001

Solution structure of the mEGF/TGFalpha44-50 chimeric growth factor.

The solution structure of the growth factor chimera mEGF/TGFalpha44-50 has been determined using an extended version of the dyana procedure for calculating structures from NMR data. The backbone fold and preferred orientation of the domains of the chimera are similar to those found in previous studies of EGF structures, and several H-bonds used as input constraints in those studies were found independently in the chimera. This shows that the modified activity of the chimera does not result from a major structural change. However, the improved precision of the structure presented here allows the origin of some unusual chemical shifts found in all of these compounds to be explained, as well as the results obtained from some site-specific mutants. Further studies of the properties of this chimeric growth factor should help to elucidate the mechanism(s) of hetero- and homodimerization of the c-erbB receptors.

Animals↗

PubMed2001

Regional effects of voluntary exercise on cell size and contraction-frequency responses in rat cardiac myocytes.

A model of voluntary exercise, in which rats are given free access to a running wheel over a 14-week period, led to left ventricular hypertrophy. To test whether the hypertrophic response to exercise was uniformly distributed across the ventricular wall, single ventricular myocytes were isolated from the sub-epicardium (EPI) and sub-endocardium (ENDO) of exercised rats and from sedentary rats for comparison. Cellular hypertrophy (approximately 20 % greater cell volume) was seen in ENDO cells from exercised animals, but no significant changes were observed in EPI cells when compared with sedentary controls. This regional effect of exercise may be a response to transmural changes in ventricular wall stress and/or strain. Cell contraction was measured as cell shortening in ENDO and EPI cells at stimulation frequencies between 1 and 9 Hz at 37 degrees C. Exercise training had no effect on cell shortening. Positive and negative contraction-frequency relationships (CFRs) were found in both EPI and ENDO cells between 1 and 5 Hz; at higher frequencies (5-9 Hz), all myocytes displayed a negative CFR. The CFR of a myocyte was, therefore, independent of regional origin and unaffected by exercise. These results suggest that, in vivo, the rat heart displays a negative CFR. We conclude that increased cell size may be a more important adaptive response to exercise than a modification of excitation-contraction coupling.

Animals↗

PubMed2000

Structural basis for the network of functional cooperativities in cytochrome c(3) from Desulfovibrio gigas: solution structures of the oxidised and reduced states.

Cytochrome c(3) is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy transduction mechanism made possible by a complex network of functional cooperativities between redox and redox/protolytic centres (the redox-Bohr effect), which enables cytochrome c(3) to work as a proton activator. The three-dimensional structures of the oxidised and reduced Desulfovibrio gigas cytochrome c(3) in solution were solved using 2D (1)H-NMR data. The reduced protein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein structure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volume restraints for paramagnetic leakage, and angle restraints derived from (13)C Fermi contact shifts of haem moiety substituents were used for the axial histidine ligands. Despite the reduction of the NOE intensities by paramagnetic relaxation, the final family of structures is of similar precision and accuracy to that obtained for the reduced form. Comparison of the two structures shows that, although the global folds of the two families of structures are similar, significant localised differences occur upon change of redox state, some of which could not be detected by comparison with the X-ray structure of the oxidised state: (1) there is a redox-linked concerted rearrangement of Lys80 and Lys90 that results in the stabilisation of haem moieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, enabling a two-electron step adapted to the function of cytochromes c(3) as the coupling partner of hydrogenase; and (2) the movement of haem I propionate 13 towards the interior of the protein upon reduction explains the positive redox-Bohr effect, establishing the structural basis for the redox-linked proton activation mechanism necessary for energy conservation, driving ATP synthesis.

Allosteric Regulation↗

PubMed2000

High yield of Methylophilus methylotrophus cytochrome c by coexpression with cytochrome c maturation gene cluster from Escherichia coli.

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.

Calorimetry, Differential Scanning↗