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D Sanders

Publications and source records attributed to D Sanders.

At least 73 records · Page 4Linked to original sources
PubMed1997

A kinetic model with ordered cytoplasmic dissociation for SUC1, an Arabidopsis H+/sucrose cotransporter expressed in Xenopus oocytes.

To elucidate the kinetic properties of the Arabidopsis H+/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range -180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H+ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (iHmax, iSmax for H+ and sucrose) and of the ligand concentrations eliciting half-maximal currents (KHm, KSm). The iHmax and iSmax exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the KHm and KSm decreased with increasingly negative membrane potential. Furthermore, at any given voltage, iSmax increased and KSm decreased as a function of the external H+ concentration. Eight six-state carrier models-which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier-were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton.

Animals↗
PubMed1997

Structure-function relations of variant and fragment nisins studied with model membrane systems.

Nisin, a 34 residue lantibiotic produced by strains of Lactococcus lactis subsp. lactis, exerts antimicrobial activity against Gram-positive bacteria at the cytoplasmic membrane. The structural aspects of nisin which facilitate membrane interaction and permeabilization have been investigated in planar lipid bilayers and liposomes with proteolytic fragments and site-directed variants. N-Terminal nisin fragments N1-12 and N1-20 had little effect on phospholipid mobility, on macroscopic electrical conductance, or on calcein release from liposomes. By contrast, the I30W nisin A variant induced a time-dependent reduction in lipid mobility, indicative of nisin-membrane surface interactions, as well as a decline in membrane capacitance, rise in conductance, and calcein release from liposomes. In these respects I30W nisin A is similar to native nisin. Charge substitutions were also engineered to generate K12L and H27K nisin A variants, both of which were similar to I30W nisin A with respect to an overall reduction in phospholipid mobility. While the K12L nisin A variant elicited a higher increase in membrane capacitance and electrical conductance than I30W nisin A, the H27K nisin A variant elicited weaker effects. These results point to a substantial role for intramembrane charged residues in controlling ion flow through nisin-doped membranes. Native nisin and variants elicit an enhanced release of calcein from liposomes composed of the negatively-charged phospholipids cardiolipin and phosphatidylserine, compared with phospholipid bearing no net charge, suggesting that an electrostatic attraction encourages the initial nisin-membrane association. The results are discussed in the context of other recently proposed models for nisin action.

Amino Acid Sequence↗
PubMed1997

Kinetics of high-affinity K+ uptake in plants, derived from K(+)-induced changes in current-voltage relationships. A modelling approach to the analysis of carrier-mediated transport.

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K(+)-H+ symporter, responsible for high-affinity K+ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K+ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K(+)-H+ symport-mediated current with a parallel, outward non-linear K+ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K+ concentration and the cytoplasmic K+ concentration. Symport activity is a complex function of the external K+ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K+ is in the millimolar range and the membrane depolarises. High cytoplasmic K+ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K+ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed.

Antiporters↗
PubMed1997

A controlled trial of cognitive behavioural therapy for non-cardiac chest pain.

BACKGROUND: The majority of patients presenting to cardiac clinics with chest pain who are reassured they do not have heart disease or other serious physical disorder continue to experience symptoms, worry about heart disease and restrict their activities. This randomized trial investigated the effectiveness of psychological treatment within routine cardiac care. METHODS: Consecutive patients presenting with chest pain and reassured by a cardiologist they do not have heart disease were reassessed 6 weeks later. Those with persistent limiting symptoms were offered the opportunity to participate in a trial of cognitive behavioural therapy. RESULTS: Thirty-seven subjects agreed to take part. A number of subjects were unenthusiastic about psychological intervention or, following explanation of the study, regarded further treatment as not being necessary. At 3 months there were significant differences between the treatment group and the control group on key outcome measures of symptoms, mood and activity. At 6 months there were fewer differences but significant advantages of treatment in terms of limitation of activities and worry about physical symptoms. CONCLUSION: We conclude that there is a need for 'stepped' further care following reassurance in the cardiac clinic and that cognitive behavioural treatment is effective with those with persistent disabling symptoms.

Adult↗
PubMed1997

Non-cardiac chest pain: why was a brief intervention apparently ineffective?

BACKGROUND: Patients who present with chest pain but have normal coronary angiography and who are told by their cardiologist that they do not have heart disease, have a poor symptomatic, psychological and quality of life outcome and remain concerned about a serious cause of their symptoms. They frequently complain they have not had enough information. The study aimed to test the effectiveness and acceptability of a brief psychological intervention based on cognitive behavioural principles. METHODS: Consecutive patients with chest pain and normal angiograms were assessed and invited to take part in a randomized controlled evaluation. The intervention consisted of an individualized information and discussion session by a specially trained cardiac nurse, together with a handout and cassette providing information and advice and telephone follow-up to discuss progress, answer questions and reiterate advice. RESULTS: The treatment proved to be unacceptable to some patients and there was no evidence of efficacy. CONCLUSIONS: Implications for the preparation of patients undergoing angiography and for the timing and delivery of information and advice following a negative result are discussed.

Analysis of Variance↗
PubMed1997

Seal-promoting solutions and pipette perfusion for patch clamping plant cells.

Patch-clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (G omega) seal between the membrane and the patch-clamp pipette. This can prove problematic in many plant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K+ and the Cl(-)concentration of the pipette solution had an effect on the seal formation. High pH and absence of K+ significantly promoted membrane sealing, whereas the concentration of Cl- had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell-attached patch, excised-patch and whole-cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.

Arabidopsis↗
PubMed1997

Inositol 1,4,5-trisphosphate-sensitive Ca2+ release across nonvacuolar membranes in cauliflower.

Previous studies have indicated that the vacuole represents the major inositol 1,4,5-trisphosphate (InsP3)-mobilizable Ca2+ pool in higher plants. This findings is in contrast to animal cells, in which the endoplasmic reticulum and plasma membrane constitute the dominant InsP3-sensitive membranes. We used membrane vesicles prepared from cauliflower (Brassica oleracae L.) inflorescences that were separated on continuous sucrose gradients to demonstrate that cauliflower possesses at least two distinct membrane populations that are sensitive to InsP3. One of these membrane populations in nonvacuolar in origin and relies upon a Ca(2+)-ATPase to accumulate Ca2+. In addition, we have shown that two polyclonal antibodies, raised against peptides corresponding to the animal type 1 InsP3 receptor, recognize immunologically related proteins in cauliflower, and that the distribution of immunoreactive proteins on a linear sucrose gradient reinforces the notion that cauliflower contains more than one membrane subtype that is sensitive to InsP3. To our knowledge, this is the first report describing an InsP3-sensitive Ca2+ store other than the vacuole in higher plant cells.

Amino Acid Sequence↗
PubMed1997

The state of neoliberalism in South Africa: economic, social, and health transformation in question.

Recent overhauls of the South African government's ruling machinery in the context of an ever-deepening commitment to neoliberal economic philosophy, have done serious, even irreparable harm to this country's political transformation. Notwithstanding some progress in policies adopted by the Department of Health, the March 1996 closure of the Reconstruction and Development Ministry and the subsequent announcement of a neoliberal macroeconomic policy have been cause for disgruntlement by those advocating progressive social and health policies.

Cost-Benefit Analysis↗
PubMed1997

Growth monitoring--is it a task worth doing in South Africa?

Childhood undernutrition is a serious public health problem in South Africa. Growth monitoring (GM) is a central tool in attempts to prevent and detect undernutrition. Despite its widespread adoption by international and national agencies, there has been very little evidence which shows that it has made an impact in reducing undernutrition. An important problem has been the conceptual confusion as to the exact role of GM. This article reviews the international evidence for the effectiveness of GM when used in various roles and proposes some of the factors necessary for GM to be successful in South Africa.

Child↗
PubMed1996

Pharmacology of Ca2+ release from red beet microsomes suggests the presence of ryanodine receptor homologs in higher plants.

Cyclic ADP-ribose (cADPR) is known to release Ca2+ from plant vacuoles, implying that this NAD+ metabolite may possess a second messenger role in plants. The degree to which the plant cADPR-gated Ca2+ release mechanism resembles cADPR action in animals has been evaluated. cADPR-elicited Ca2+ release from red beet microsomes was inhibited by 1 mM procaine but insensitive to heparin. Furthermore, pre-release of Ca2+ from red beet vesicles by either 5 mM caffeine or micromolar levels of ryanodine precluded further Ca2+ mobilisation by cADPR. Thus, this study argues strongly for conservation between the plant and animal cADPR-elicited Ca2+ release mechanisms.

Adenosine Diphosphate Ribose↗
PubMed1996

Interaction of the lantibiotic nisin with membranes revealed by fluorescence quenching of an introduced tryptophan.

Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of gram-positive bacteria. To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue. Activity of the site-directed variant containing tryptophan was established to be similar to that of the wild-type peptide. Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment. Quenching experiments with aqueous and membrane-restricted quenchers (iodide and spin-labelled lipids, respectively) both confirmed a non-aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity. The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles. This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes.

Amino Acid Sequence↗
PubMed1996

Water-soluble, recombinant CuA-domain of the cytochrome ba3 subunit II from Thermus thermophilus.

Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm(-1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study.

Amino Acid Sequence↗
PubMed1996

Effect of DNA conformation on cisplatin adduct formation.

The anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) has been shown previously to form adducts preferentially within internucleosomal or linker DNA rather than to DNA within the nucleosome. To determine whether other "open" regions of chromatin have an increased affinity for cisplatin, adduct formation within specific chromatin domains was analyzed. There was a significant increase in cisplatin-DNA adduct formation for DNA associated with the nuclear matrix (NM) compared with other chromatin domains and total unfractionated DNA. In contrast, treatment of the same cells with trans-diamminedichloroplatinum(II) (transplatin) did not result in preferential adduct formation. These findings led to the hypothesis that it might be possible to alter DNA to make it a more favorable target for cisplatin. The effect of arginine butyrate on cisplatin-DNA adduct formation was analyzed in human cancer cells. The combination of arginine butyrate and cisplatin resulted in a concentration-responsive increase in cisplatin-DNA adduct formation in PC-3 cells and an overall increase in cisplatin-DNA adduct formation in three other human cancer cell lines. The same combination also resulted in a significant increase in drug-induced cytotoxicity at a low concentration of cisplatin. These results suggest that chromatin configuration can affect cisplatin adduct formation.

Antineoplastic Agents↗
PubMed1996

Reaction kinetics of the vacuolar H(+)-pumping ATPase in beta vulgaris.

Vacuolar-type H(+)-ATPases (V-ATPases) are ubiquitous in eukaryote endomembranes, where they are responsible for lumenal acidification. The ratios of H+ translocated per ATP hydrolyzed (which may be important in controlling the activity of these pumps) have previously been found to be variable, noninteger and sensitive to cytosolic as well as lumenal pH. The mechanistic implications of these findings are explored here with reaction kinetics. Experimental data for this analysis comprise supra- and superlinear V-ATPase current-voltage relationships, isolated as bafilomycin-sensitive currents in vacuolar membranes from Beta using the "whole vacuole" patch clamp configuration. Whereas simple models with one reaction cycle fail to provide an adequate description of the data (mainly because of a weak sensitivity of the zero-current voltage to the transmembrane pH gradient), a model with two linked reaction loops allowing partial coupling of H+ translocation to ATP hydrolysis does provide good descriptions. All experimental data obtained with the same vacuolar pH (4.3) could be reduced to a model with eleven independent parameters. Best fits have been obtained on the basis of a binding domain possessing 3 H+ binding sites per ATP hydrolyzed and a net charge of -2 when unoccupied. The enzyme could reorientate its access site between the vacuolar and cytoplasmic side when zero, one or three H+ are bound.

In Vitro Techniques↗