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D Sanders

Publications and source records attributed to D Sanders.

At least 55 records · Page 3Linked to original sources
PubMed1999

Selenomethionine-substituted Thermus thermophilus cytochrome ba3: characterization of the CuA site by Se and Cu K-EXAFS.

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA. Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.

Amino Acid Sequence↗
PubMed1999

Plasma membrane transport in context - making sense out of complexity.

Major advances in our understanding of the transport of inorganic nutrient ions across plant plasma membranes have emerged from recent studies on the control of the dominant H+-pumping ATPase and from identification of a range of new transporters for divalent cations, potassium, phosphate and nitrate. In many cases, multiple transporter isoforms have been described. An appreciation of the physiological roles of these transporters demands combined genetic and physiological approaches, which, in the case of an outward rectifying K+ channel, have already been used to yield an intriguing insight into root-mediated K+ release into the xylem. In this review we attempt to place some of those developments in a physiological context.

Biological Transport↗
PubMed1999

Functional characterization of N-methyl-D-aspartic acid-gated channels in bone cells.

Our recent identification of glutamate receptors in bone cells suggested a novel means of paracrine communication in the skeleton. To determine whether these receptors are functional, we investigated the effects of the excitatory amino acid, glutamate, and the pharmacological ligand, N-methyl-D-aspartic acid (NMDA), on glutamate-like receptors in the human osteoblastic cell lines MG63 and SaOS-2. Glutamate binds to osteoblasts, with a Kd of approximately 10(-4) mol/L and the NMDA receptor antagonist, D(L)-2-amino-5-phosphonovaleric acid (D-APV), inhibits binding. Using the patch-clamp technique, we measured whole-cell currents before and after addition of L-glutamate or NMDA and investigated the effects of the NMDA channel blockers, dizolcipine maleate (MK801), and Mg2+, and the competitive NMDA receptor antagonist, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid (R-CPP), on agonist-induced currents. Both glutamate and NMDA induced significant increases in membrane currents. Application of Mg2+ (200 micromol/L) and MK801 (100 micromol/L) caused a significant decrease in inward currents elicited in response to agonist stimulation. The competitive NMDA receptor antagonist, R-CPP (100 micromol/L), also partially blocked the NMDA-induced currents in MG63 cells. This effect was reversed by addition of further NMDA (100 micromol/L). In Fura-2-loaded osteoblasts, glutamate induced elevation of intracellular free calcium, which was blocked by MK801. These results support the hypothesis that glutamate plays a role in bone cell signaling and suggest a possible role for glutamate agonists/antagonists in the treatment of bone diseases.

Binding Sites↗
PubMed1999

The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution.

The structure of the CuA-containing, extracellular domain of Thermus thermophilus ba3-type cytochrome c oxidase has been determined to 1.6 A resolution using multiple X-ray wavelength anomalous dispersion (MAD). The Cu2S2 cluster forms a planar rhombus with a copper-copper distance of 2.51 +/- 0.03 A. X-ray absorption fine-structure (EXAFS) studies show that this distance is unchanged by crystallization. The CuA center is asymmetrical; one copper is tetrahedrally coordinated to two bridging cysteine thiolates, one histidine nitrogen and one methionine sulfur, while the other is trigonally coordinated by the two cysteine thiolates and a histidine nitrogen. Combined sequence-structure alignment of amino acid sequences reveals conserved interactions between cytochrome c oxidase subunits I and II.

Amino Acid Sequence↗
PubMed1999

Histologic and physiologic effects of electrocautery, CO2 laser, and radiofrequency injury in the porcine soft palate.

OBJECTIVES: This study was undertaken to compare the effects of electrosurgery, CO2 laser, and radiofrequency on the histological structure and physiological properties of the soft palate. These surgical techniques are used for stiffening and reducing the soft palate in the treatment of snoring and mild sleep apnea. STUDY DESIGN: Fifteen pigs were divided into five groups. METHODS: group 1 underwent electrosurgery incision of the lateral soft palate and excision of the uvula. Group 2 underwent the same procedure with the CO2 laser. Group 3 underwent CO2 laser ablation of the midline soft palate mucosa. Group 4 underwent radiofrequency volumetric reduction. Group 5 served as control. After 5 weeks the animals were sacrificed. Soft palate mucosa and muscle were subjected to tensiometric and histological analysis. Statistical analysis of tensiometric measurements was done with an ANOVA using a Bonferroni-Dunn correction. RESULTS: Tensiometric measurements of the mucosa were increased over normal controls when the surgical technique resulted in mucosal disruption. When the surgical technique resulted in primarily muscle disruption (radiofrequency) tensiometric measurements of the muscle were increased over controls. Histological analysis demonstrated normal remucosalization in all specimens, with fibrosis increased at the site of the primary injury. CONCLUSIONS: The type of injury produced resulted in identifiable patterns of physiological and histological change. Understanding of the effects of available surgical techniques is important in guiding our choice of surgical approach and allows us to better counsel our patients on their surgical options.

Animals↗
PubMed1999

K+-Selective inward-rectifying channels and apoplastic pH in barley roots

Recent structure-function analysis of heterologously expressed K+-selective inward-rectifying channels (KIRCs) from plants has revealed that external protons can have opposite effects on different members of the same gene family. An important question is how the diverse response of KIRCs to apoplastic pH is reflected at the tissue level. Activation of KIRCs by acid external pH is well documented for guard cells, but no other tissue has yet been studied. In this paper we present, for the first time to our knowledge, in planta characterization of the effects of apoplastic pH on KIRCs in roots. Patch-clamp experiments on protoplasts derived from barley (Hordeum vulgare) roots showed that a decrease in external pH shifted the half-activation potential to more positive voltages and increased the limit conductance. The resulting enhancement of the KIRC current, together with the characteristic voltage dependence, strongly relates the KIRC of barley root cells to AKT1-type as opposed to AKT3-type channels. Measurements of cell wall pH in barley roots with fluorescent dye revealed a bulk apoplastic pH close to the pK values of KIRC activation and significant acidification of the apoplast after the addition of fusicoccin. These results indicate that channel-mediated K+ uptake may be linked to development, growth, and stress responses of root cells via the activity of H+-translocating systems.

Journal Article↗
PubMed1999

Divalent cation block of inward currents and low-affinity K+ uptake in Saccharomyces cerevisiae.

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Delta trk2Delta strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K+ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K+ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K+ with Rb+, Cs+, or Na+ only slightly modulated the magnitude of the inward current, whereas replacement with Li+ reduced the inward current by approximately 50%, and tetraethylammonium (TEA+) and choline were relatively impermeant. The inward current was blocked by extracellular Ca2+ and Mg2+ with apparent Kis (at -140 mV) of 363 +/- 78 and 96 +/- 14 microM, respectively. Furthermore, decreasing cytosolic K+ increased the magnitude of the inward current independently of the electrochemical driving force for K+ influx, consistent with regulation of the inward current by cytosolic K+. Uptake of 86Rb+ by intact trk1Delta trk2Delta cells was inhibited by extracellular Ca2+ with a Ki within the range observed for the inward current. Furthermore, increasing extracellular Ca2+ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.

Calcium↗
PubMed1998

Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose.

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5'-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ >/= 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126-2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.

Journal Article↗
PubMed1998

High-potential states of blue and purple copper proteins.

Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA). In each case, the high-potential state forms reversibly. Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein. It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein.

Azurin↗
PubMed1998

Cloning and expression in Escherichia coli of the cytochrome c552 gene from Thermus thermophilus HB8. Evidence for genetic linkage to an ATP-binding cassette protein and initial characterization of the cycA gene products.

We report sequence of Thermus thermophilus HB8 DNA containing the gene (cycA) for cytochrome c552 and a gene (cycB) encoding a protein homologous with one subunit of an ATP-binding cassette transporter. The cycA gene encodes a 17-residue N-terminal signal peptide with following amino acid sequence identical to that reported by (Titani, K., Ericsson, L. H., Hon-nami, K., and Miyazawa, T. (1985) Biochem. Biophys. Res. Commun. 128, 781-787). A modified cycA was placed under control of the T7 promoter and expressed in Escherichia coli. Protein identical to that predicted from the gene sequence was found in two heme C-containing fractions. Fraction rC552, characterized by an alpha-band at 552 nm, contains approximately 60-70% of a protein highly similar to native cytochrome c552 and approximately 30-40% of a protein that contains a modified heme. Cytochrome rC552 is monomeric and is an excellent substrate for cytochrome ba3. Cytochrome rC557 is characterized by an alpha-band at 557 nm, contains approximately 90% heme C and approximately 10% of non-C heme, exists primarily as a homodimer, and is essentially inactive as a substrate for cytochrome ba3. We suggest that rC557 is a "conformational isomer" of rC552 having non-native, axial ligands to the heme iron and an "incorrect" protein fold that is stabilized by homodimer formation.

Amino Acid Sequence↗
PubMed1998

Effect of redox state on the folding free energy of a thermostable electron-transfer metalloprotein: the CuA domain of cytochrome oxidase from Thermus thermophilus.

The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide.

Copper↗
PubMed1998

Cell marking in Arabidopsis thaliana and its application to patch-clamp studies.

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch-clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch-clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch-clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch-clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch-clamp studies.

Arabidopsis↗
PubMed1998

Zimbabwe's hospital referral system: does it work?

BACKGROUND: Anecdotal evidence has suggested inefficiency in the pyramidal health care referral system established in Zimbabwe in 1980, as part of its primary health care (PHC) model. AIM: To assess the functioning of the pyramidal referral system in two rural districts surrounding Harare, Zimbabwe, with regard to two common indicator conditions: pneumonia in children and malaria in adults. METHODS: For a three-month period, all complete inpatient records with discharge diagnoses of pneumonia or malaria from three hospitals representing different levels of care were analyzed (n = 227). Data were collected on demographic and patient care variables. The appropriateness of admissions and referrals was determined by an assessment of the severity of illness and 'intensiveness' of care required. Data were analyzed for differences among the three hospitals and between the two indicator conditions. Per night inpatient bed costs for each hospital were also calculated. RESULTS: For pneumonia in children, 56.8% of patients admitted at the secondary level, 53.8% of patients at the tertiary level and 57.8% of patients at the quaternary level were of mild severity. For malaria in adults, 74.0% of patients seen at the secondary level, 81.5% of patients at the tertiary level and 54.3% at the quaternary level were of mild severity. For pneumonia, were no differences in severity between the three hospitals whereas for malaria significant case-mix differences among the hospitals were found. Most patients attending the highest level referral facility were inappropriate admissions who could have been treated at a lower level of care. The majority of patients at all the hospitals studied had used that hospital as their first or second point of contact with the health services. There were large variations in the inpatient per night bed costs between the three hospitals. CONCLUSIONS: Using the indicator diseases of pneumonia in children and malaria in adults, this study concluded that this network did not meet design expectations as the central level referral hospital cared for a similar case-mix of patients as the district level, but at six times the cost. The appropriateness of admissions and referrals could be improved by developing or strengthening intermediate level facilities, by changing mechanisms of access to specialist facilities and by training health professionals in community settings.

Adult↗
PubMed1998

Control of Cl- efflux in chara corallina by cytosolic pH, free ca2+, and phosphorylation indicates a role of plasma membrane anion channels in cytosolic pH regulation

Enhanced Cl- efflux during acidosis in plants is thought to play a role in cytosolic pH (pHc) homeostasis by short-circuiting the current produced by the electrogenic H+ pump, thereby facilitating enhanced H+ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pHc activates Cl- efflux via two mechanisms. The first is a direct effect of pHc on Cl- efflux; the second mechanism comprises a pHc-induced increase in affinity for cytosolic free Ca2+ ([Ca2+]c), which also activates Cl- efflux. Cl- efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pHc and [Ca2+]c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl- efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl- efflux at 60% of the maximal level in a manner that was both pHc and [Ca2+]c independent. These findings imply that plasma membrane anion channels play a central role in pHc regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction.

Journal Article↗
PubMed1997

The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating.

The yeast Saccharomyces cerevisiae gene CCH1 (ORF YGR217w) shows high homology with animal calcium channel alpha1-subunit genes. Knock-out mutants were constructed of Cch1 and of Mid1 which is known to mediate Ca2+ influx in response to the alpha-mating pheromone. Cch1 is involved in calcium influx and the late stage of the mating process. The cch1 mutant sensitivity against the alpha-mating pheromone can be reduced by the addition of extra calcium. The product of this gene is likely to interact with the MID1 gene product in Ca influx or its control.

Base Sequence↗