New powered wheelchair systems for the rehabilitation of some severely disabled users.
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Persons with Disabilities↗
Biomedical subjects
D Sanders
Publications and source records attributed to D Sanders.
The use of virtual reality to train powered wheelchair users and test new wheelchair systems.
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Algorithms↗
Differential ion accumulation and ion fluxes in the mesophyll and epidermis of barley.
In barley (Hordeum vulgare L.) leaves, differential ion accumulation commonly results in inorganic phosphate (Pi) being confined to the mesophyll and Ca(2+) to the epidermis, with preferential epidermal accumulation of Cl(-), Na(+), and some other ions. The pattern was confirmed in this study for major inorganic anions and cations by analysis of barley leaf protoplasts. The work focused on the extent to which differences in plasma membrane ion transport processes underlie these observations. Ion transport across the plasma membrane of barley epidermal and mesophyll protoplasts was investigated electrophysiologically (by microelectrode impalement and patch clamping) and radiometrically. Data from both approaches suggested that similar types of ion-selective channels and membrane transporters, which catalyze the transport of Ca(2+), K(+), Na(+), and Pi, exist in the plasma membrane of the two cell types. In general, the simple presence or absence of ion transporters could not explain cell-type-specific differences in ion accumulation. However, patch-clamp data suggested that differential regulation of instantaneously activating ion channels in the plasma membrane could explain the preferential accumulation of Na(+) in the epidermis.
Calcium↗
Sodium-dependent nitrate transport at the plasma membrane of leaf cells of the marine higher plant Zostera marina L.
NO(3)(-) is present at micromolar concentrations in seawater and must be absorbed by marine plants against a steep electrochemical potential difference across the plasma membrane. We studied NO(3)(-) transport in the marine angiosperm Zostera marina L. to address the question of how NO(3)(-) uptake is energized. Electrophysiological studies demonstrated that micromolar concentrations of NO(3)(-) induced depolarizations of the plasma membrane of leaf cells. Depolarizations showed saturation kinetics (K(m) = 2.31 +/- 0.78 microM NO(3)(-)) and were enhanced in alkaline conditions. The addition of NO(3)(-) did not affect the membrane potential in the absence of Na(+), but depolarizations were restored when Na(+) was resupplied. NO(3)(-)-induced depolarizations at increasing Na(+) concentrations showed saturation kinetics (K(m) = 0.72 +/- 0.18 mM Na(+)). Monensin, an ionophore that dissipates the Na(+) electrochemical potential, inhibited NO(3)(-)-evoked depolarizations by 85%, and NO(3)(-) uptake (measured by depletion from the external medium) was stimulated by Na(+) ions and by light. Our results strongly suggest that NO(3)(-) uptake in Z. marina is mediated by a high-affinity Na(+)-symport system, which is described here (for the first time to our knowledge) in an angiosperm. Coupling the uptake of NO(3)(-) to that of Na(+) enables the steep inwardly-directed electrochemical potential for Na(+) to drive net accumulation of NO(3)(-) within leaf cells.
Cell Membrane↗
Understanding mail survey response rates among male reserve component Gulf War era veterans.
In this study of current and former male Reserve and National Guard members from the State of Ohio, it was expected that veterans who were older, had more years of military service, who had participated in the Persian Gulf War, who were Euro-Americans, who were higher in rank, who had higher residential stability in Ohio, who belonged to the Air Force, who had higher formal education, and who belonged to the National Guard would have a greater investment in U.S. society as defined by 11 demographic variables. It was assumed that those with greater investment in society would more often have valid addresses and would be more likely to respond to a survey on military issues, thereby biasing sample outcomes in those directions. Results for male veterans were consistent with the hypothesis that investment in the society system would predict validity of addresses and response rates. In other words, results supported the idea that those veterans who might be expected to have a greater investment in U.S. society were more likely to be located and to respond (once located) to a survey concerning Desert Storm-era military service and its aftermath. Implications for future Desert Storm research are discussed.
Adult↗
Assay for free and total carnitine in human plasma using tandem mass spectrometry.
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Acetylcarnitine↗
Calcium-induced calcium release mediated by a voltage-activated cation channel in vacuolar vesicles from red beet.
Little is known about the mechanisms underlying calcium-induced Ca2+ release (CICR) in plants. The slow-activating vacuolar (SV) channel is both permeable to, and activated by Ca2+, and is therefore a prime candidate for a role in CICR. Cytosol-side-out vacuolar membrane vesicles loaded with 45Ca2+ showed voltage- and Ca(2+)-dependent Ca2+ release, which was sensitive to the SV channel modulators DIDS, protein phosphatase 2B and calmodulin. Significantly, voltage-dependent Ca2+ release strongly depended on cytoplasmic Ca2+ concentrations. The results support the notion that CICR occurs in plant cells and that the process can be catalysed by the SV channel on the vacuolar membrane.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
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↗
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↗
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↗
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↗
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↗
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↗
Communicating with calcium
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Journal Article↗
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↗
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↗
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↗
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↗