Biomedical subjects

W J Lucas

Publications and source records attributed to W J Lucas.

At least 55 records · Page 3Linked to original sources

PubMed1992

Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA.

Inward-rectifying potassium channels located in the plasma membrane of higher plant and animal cells contribute to cellular homeostasis and excitability. The genes encoding this specific class of K+ channels have not been functionally identified. This report shows that a single messenger RNA transcript from the Arabidopsis thaliana KAT1 complementary DNA confers the functional expression of a hyperpolarization-activated K+ channel in Xenopus oocytes. The channels encoded by KAT1 are highly selective for K+ over other monovalent cations, are blocked by tetraethylammonium and barium, and have a single channel conductance of 28 +/- 7 picosiemens with 118 millimolar K+ in the bathing solution. These functional characteristics, typical of inward-rectifying K+ channels in eukaryotic cells, demonstrate that KAT1 encodes an inward-rectifying K+ channel.

Animals↗

PubMed1992

Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae.

We report the isolation of a cDNA (KAT1) from Arabidopsis thaliana that encodes a probable K+ channel. KAT1 was cloned by its ability to suppress a K+ transport-defective phenotype in mutant Saccharomyces cerevisiae cells. This suppression is sensitive to known K+ channel blockers, including tetraethylammonium and Ba2+ ions. The KAT1 cDNA contains an open reading frame capable of encoding a 78-kDa protein that shares structural features found in the Shaker superfamily of K+ channels. These include a cluster of six putative membrane-spanning helices (S1-S6) at the amino terminus of the protein, a presumed voltage-sensing region containing Arg/Lys-Xaa-Xaa-Arg/Lys repeats within S4, and the highly conserved pore-forming region (known as H5 or SS1-SS2). Our results suggest that the structural motif for K+ channels has been conserved between plants and animals.

Amino Acid Sequence↗

PubMed1992

Reaction kinetic model of a proposed plasma membrane two-cycle H(+)-transport system of Chara corallina.

Biophysical and numerical analysis methods were used to characterize and model the transport protein that gives rise to the acid and alkaline regions of Chara. A measuring system that permits the detection of area-specific current-voltage curves was used. These current-voltage curves, obtained from the inward current regions of Chara, underwent a parallel shift when the alkaline region was inverted by means of an acid pH treatment. In this situation the reversal potential of this area shifted from -120 mV to -340 mV. Together with data obtained from experiments using a divided chamber system, these results suggest that a common transport protein generates inward and outward current regions of Chara. On the basis of these experimental findings, a reaction kinetic model is proposed that assigns two operational modes to the proposed transport protein. Switching between these modes generates either acid or alkaline behavior. Since the observed pH dependence of the postulated transporter is rather complex, a reaction kinetic saturation mechanism had to be incorporated into the model. This final 10-state reaction kinetic model provides an appropriate set of mathematical relations to fit the measured current-voltage curves by computer.

Carrier Proteins↗

PubMed1992

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.

Cytoplasm↗

PubMed1991

Altered function of the tobacco mosaic virus movement protein in a hypersensitive host.

The N gene in Nicotiana sp. confers hypersensitive resistance to all strains of tobacco mosaic virus (TMV) and limits the rate of virus spread in infected leaves. To examine the role of the movement protein (MP) of TMV in the hypersensitive reaction (HR), transgenic Nicotiana tabacum cv. Xanthi-nc (genotype NN) plants that express the MP gene were produced and the molecular size exclusion limit of plasmodesmata in leaf mesophyll cells was monitored. At the HR-permissive temperature (24 degrees) movement from cell to cell of fluorescein isothiocyanate-labeled dextran of molecular mass 3.9 kDa was detected while 9.4-kDa molecules failed to move. At the HR-nonpermissive temperature (33 degrees) the 9.4-kDa probe moved readily from cell to cell. In contrast, in transgenic Xanthi (genotype nn) which express the MP gene the 9.4-kDa probe moved from cell to cell at 24 and 33 degrees. These results suggest that the N gene may modify the ability of the MP to alter plasmodesmata molecular exclusion limits, although expression of the TMV-MP gene alone did not induce the HR. Furthermore, when MP(+) Xanthi-nc tobacco lines were inoculated with a TMV that lacked a MP gene the HR was induced, and the concentration of MP in the transgenic lines was correlated with the degree of the HR.

Capsid Proteins↗

PubMed1991

The TMV movement protein: role of the C-terminal 73 amino acids in subcellular localization and function.

The role of the C-terminal one-third of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP) on its subcellular localization and on virus spread was investigated. We have constructed eight cDNAs encoding MPs with variable size deletions from the C-terminal end. Expression of the truncated proteins was verified in recombinant yeast using an antiserum directed to a synthetic peptide corresponding to 21 amino acids near the N-terminal end of the MP. In transgenic tobacco plants, MP from which more than 55 amino acids were deleted no longer accumulated in the cell wall fraction of a cellular extract, where the complete MP accumulates. Dye diffusion studies showed that both unmodified and modified MPs that accumulate in the cell wall fraction are able to alter plasmodesmatal size exclusion limits. Biological function of the modified MPs was tested in the transgenic plants with the TMV thermosensitive mutant Ls1 and a TMV genomic RNA transcript lacking a functional MP. There was a correlation between the cell wall localization of the modified MPs and its ability to potentiate virus spread. The results presented here demonstrate the dispensability of the C-terminal 55 amino acids of the MP in its subcellular localization in tobacco plants and its role in virus movement. Moreover, our results show that a stretch of 19 amino acids (195 to 213) is essential for localization of the MP to the cell wall fraction of plant cells.

Capsid↗

PubMed1991

Autonomous Local Area Control over Membrane Transport in Chara Internodal Cells.

Internodal cells of Chara were separated with a Plexiglas divider into two segments and the vibrating probe was used to investigate the extracellular current profiles that formed along these two surfaces. Treating one segment of the Chara cell with K(+) concentrations greater than 2 millimolar caused a dramatic reduction in the extracellular current pattern in this compartment. Concentrations of 5, 10, and 20 millimolar K(+) were used to establish that a normal current profile could be maintained along the cell surface in the control compartment, whereas the extracellular current profile was strongly reduced along the entire cell surface that was located in the second, high-K(+) compartment. Simultaneous measurements of the membrane potential in the two segments of the divided Chara cell established that, in the presence of elevated K(+) concentrations, a longitudinal voltage gradient of up to 60 millivolts was maintained. Experiments in which the pH value in one compartment was either decreased (pH 6.0) or increased (pH 11) gave rise to a reduced extracellular current profile along this segment of the cell, whereas the pattern in the control segment remained unaltered. These results are discussed in terms of the cellular spatial control system that must function to regulate the regions of outward and inward current, and the concept of autonomous local area (domain) control is presented.

Journal Article↗

PubMed1991

Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein.

A gene encoding a temperature-sensitive mutant (MPP154A) of the 30-kilodalton movement protein (MP) of tobacco mosaic virus (TMV) was transformed into Nicotiana tabacum cv Xanthi. Transgenic plants expressing the MPP154A gene complemented local and systemic movement of an MP-defective mutant of TMV (U3/12MPfs) at the permissive temperature of 24 degrees C but not at 32 degrees C, the nonpermissive temperature. A microinjection procedure was used to investigate the effects of the modified TMV MP on plasmodesmatal size-exclusion limits. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran), with an average molecular mass of 9.4 kilodaltons, was detected between leaf mesophyll cells of the transgenic plants at 24 degrees C; however, no movement of either 3.9-kilodalton or 9.4-kilodalton F-dextrans was detected when the transgenic plants were held for 6 hours (or longer) at 32 degrees C. When these plants were shifted back to 24 degrees C for 6 hours, cell-to-cell movement of the F-dextrans was again observed. Accumulation of MPP154A was not affected by the temperature regime, nor was the subcellular distribution of the MP altered. These results are consistent with a change in the protein conformation of MPP154A at the nonpermissive temperature, which gives rise to a protein that fails to modify the molecular size-exclusion limits of plasmodesmata to the same extent as wild-type MP. Surprisingly, at 32 degrees C, movement of the F-dextrans was inhibited in transgenic plants expressing the wild-type MP gene; however, the inhibition was transient and was no longer detected after 48 hours at this elevated temperature. This transient inhibition of plasmodesmatal function was alleviated with Sirofluor, an inhibitor of callose ([1----3]-beta-D-glucan) synthesis. This result provides experimental evidence that callose deposition is involved in regulating the molecular size-exclusion limit of plasmodesmata in plants. Sirofluor had no effect on the inhibition of F-dextran movement at 32 degrees C in plants expressing the MPP154A gene, indicating that callose formation was not responsible for the failure of the temperature-sensitive mutant protein to alter the size-exclusion limit of plasmodesmata.

Benzenesulfonates↗

PubMed1990

Inversion of extracellular current and axial voltage profile in Chara and Nitella.

Reducing the pH of the bathing solution from 8.2 to pH 6 can induce an inversion of the extracellular current pattern that develops at the surface of Chara corallina internodal cells. A similar result can be obtained on some cells by changing the medium to a pH value of 10. In noninverting Chara cells the currents were strongly reduced when the pH value of the medium was changed between 3 and 11. Simultaneous measurements of the Chara transmembrane potential in the acid and alkaline regions revealed that a light-induced electrical potential gradient of approximately 24 mV was present in the axial (or longitudinal) direction. Correlated to the external current pattern inversion was an inversion of this internal longitudinal voltage gradient. Reillumination of Nitella cells, after a period of darkness, often resulted in a complete inversion of the extracellular current pattern. These results are discussed in terms of spatial and temporal control of membrane transport processes, and in particular the control of current loops that pass through these cells.

Chlorophyta↗

PubMed1990

Advances in oral anti-arrhythmic therapy: implications for the anaesthetist.

Surgical patients often are receiving antiarrhythmic therapy. Thus, because anaesthetic agents can affect cardiac function and may interact with concurrent antiarrhythmic medications, the anaesthetist should be aware of the electrophysiology associated with dysrhythmias and their management. Tocainide, flecainide, mexiletine, encainide and amiodarone have been introduced recently and each has an unique pattern of bioavailability, metabolism and toxicity. Patients treated with these drugs need special concern as they have abnormal cardiovascular systems and may be at increased risk for perioperative morbidity. In addition, unexpected untoward reactions and toxicity can result from interactions of anaesthetic agents and these drugs. This review discusses normal cardiac electrophysiology, common dysrhythmias and the electrophysiological effects of the newer oral antiarrhythmic drugs.

Administration, Oral↗

PubMed1990

Molecular characterization and biological function of the movement protein of tobacco mosaic virus in transgenic plants.

We previously demonstrated, in transgenic tobacco plants, that the role of the movement protein (MP) of tobacco mosaic virus is to facilitate the cell-to-cell spread of viral progeny during infection. An analysis of different tissues of these transgenic plants indicated that the MP accumulated in leaf, stem, and root tissue. The highest levels were detected in older leaves. The relative levels of MP in leaf tissue from transgenic plants were equivalent to, or higher than, the levels of MP in tobacco mosaic virus-infected leaf tissue. Results of subcellular fractionation of homogenates of transgenic leaf tissue showed that the MP was most abundant in the cell wall fraction of older leaves and that the protein remained at high levels in the cell wall fraction as the leaves continued to age. Significant levels of the MP were detected in a crude membrane/organelle fraction and a soluble fraction in younger leaves but decreased to low levels in older leaves. These results suggest that the MP accumulates and is stable in cell walls. We have previously shown that the MP modifies the molecular exclusion limit of plasmodesmata, which is consistent with the hypothesis that plant viruses move from cell to cell through altered plasmodesmata. We show here that the ability of the tobacco mosaic virus MP to modify the molecular exclusion limit of plasmodesmata in tobacco depends on the developmental stage of the leaf. The implications of these findings on understanding virus movement and how plasmodesmata function are discussed.

Capsid Proteins↗

PubMed1989

Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.

The function of the 30-kilodalton movement protein (MP) of tobacco mosaic virus is to facilitate cell-to-cell movement of viral progeny in an infected plant. A novel method for delivering non-plasmalemma-permeable fluorescent probes to the cytosol of spongy mesophyll cells of tobacco leaves was used to study plasmodesmatal size exclusion limits in transgenic plants that express the MP gene. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran) with an average molecular mass of 9400 daltons and an approximate Stokes radius of 2.4 nanometers was detected between cells of the transgenic plants, whereas the size exclusion limit for the control plants was 700 to 800 daltons. No evidence of F-dextran metabolism in the leaves of the transgenic plants was found. Thus, the tobacco mosaic virus movement protein has a direct effect on a plasmodesmatal function.

Dextrans↗

PubMed1989

High affinity k uptake in maize roots: a lack of coupling with h efflux.

We report here on the putative coupling between a high affinity K(+) uptake system which operates at low external K(+) concentrations (K(m) = 10-20 micromolar), and H(+) efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K(+)((86)Rb(+)) influx, and the simultaneous measurement of net K(+) and H(+) fluxes associated with individual cells at the root surface with K(+)- and H(+)-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K(+) and H(+) ions within the unstirred layer at the root surface. No evidence for coupling between K(+) uptake and H(+) efflux could be found based on: (a) extremely variable K(+):H(+) flux stoichiometries, with K(+) uptake often well in excess of H(+) efflux; (b) dramatic time-dependent variability in H(+) extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K(+) uptake system (to changes in external pH) when net K(+) uptake, unidirectional K(+)((86)Rb(+)) influx, and K(+)-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K(+) absorption into maize root cells is not mediated by a K(+)/H(+) exchange mechanism. Instead, it is either due to the operation of a K(+)-H(+) cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K(+)-ATPase as reported for Escherichia coli and Saccharomyces.

Journal Article↗

PubMed1989

Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation.

In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.

Base Sequence↗

PubMed1988

The intra-aortic balloon pump: a review.

Intra-aortic balloon pump therapy is often life-saving in patients with refractory cardiac failure following cardiopulmonary bypass, in the peri-infarct period, or in treating complications of coronary artery angioplasty. Anesthesiologists are intimately involved in the care of the majority of IABP patients and require a thorough understanding of the equipment, its physiologic effects, and potential complications.

Animals↗

PubMed1987

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis.

An O(2) electrode system with a specially designed chamber for ;whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O(2) concentrations on photosynthetic O(2) evolution. At pH = 5.5 and 20% O(2), cells grown in HCO(3) (-) medium (low CO(2), pH >/= 9.0) exhibited a higher affinity for external CO(2) (K((1/2))(CO(2)) = 40 +/- 6 micromolar) than the cells grown for at least 24 hours in high-CO(2) medium (pH = 6.5), (K((1/2))(CO(2)) = 94 +/- 16 micromolar). With O(2) </= 2% in contrast, both types of cells showed a high apparent affinity (K((1/2))(CO(2)) = 50 - 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO(2) medium (pH = 6.5). The high-pH, HCO(3) (-)-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO(2) which exceeded the CO(2) externally supplied, and the reverse situation has been observed in high-CO(2)-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO(3) (-) (K((1/2))[HCO(3) (-)]) was 0.6 +/- 0.2 millimolar, at 20% O(2). But under low O(2) concentrations (</=2%), surprisingly, an inhibition of net O(2) evolution was elicited, which was maximal at low HCO(3) (-) concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO(2) medium, (b) Chara cells are able to accumulate CO(2) internally by means of a process apparently independent of the plasmalemma HCO(3) (-) transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO(3) (-): pseudocyclic electron flow, sustained by O(2) photoreduction, may produce the additional ATP needed for the HCO(3) (-) transport.

Journal Article↗

PubMed1987

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : II. Product Inhibition by UDP.

The mode of inhibition of UDP, one of the products of the reaction catalyzed by (1-->3)-beta-d-glucan synthase in sugar beet (Beta vulgaris L.) was investigated. In the absence of added UDP, the enzyme, in the presence of Ca(2+), Mg(2+), and cellobiose, exhibited Michaelis-Menten kinetics and had an apparent K(m) of 260 micromolar for UDP-glucose. Complex effects on the kinetics of the (1-->3)-beta-d-glucan synthase were observed in the presence of UDP. At high UDP-glucose concentrations, i.e. greater than the apparent K(m), UDP behaved as a competitive inhibitor with an apparent K(i) of 80 micromolar. However, at low UDP-glucose concentrations, reciprocal plots of enzyme activity versus substrate concentration deviated sharply from linearity. This unusual effect of UDP is similar to that reported for fungal (1-->3)-beta-d-glucan synthase. However, papulacandin B, a potent inhibitor of this fungal enzyme, had no effect on the plant (1-->3)-beta-d-glucan synthase isolated from sugar beet petioles. The inhibitory effect of UDP was also compared with other known inhibitors of glucan synthases.

Journal Article↗

PubMed1987

Influence of internal sugar levels on apoplasmic retrieval of exogenous sucrose in source leaf tissue.

Sugar levels in Beta vulgaris leaves were increased by heat-girdling the petiole and returning the plant to the controlled-environment chamber for 10 and 34 hours. After 10 hours, sucrose influx into the treated leaves was similar to the controls, although sucrose levels increased from 2.1 to 5.3 micromoles per milligram chlorophyll. However, after a 34-hour treatment, sucrose levels increased from 2.1 to 11.5 micromoles per milligram chlorophyll. In this instance, sucrose influx decreased relative to the untreated controls. Decreasing sugar levels by DCMU treatment resulted in a small stimulation of sucrose influx. A similar DCMU treatment applied to leaves of Allium cepa also resulted in an increase in sucrose influx. However, in A. cepa we could not attribute this increase to a lowering of sugar levels, as the kinetic profiles obtained from control leaves did not vary from each other throughout the day, despite considerable changes in sugar levels. Additionally, it appeared that sucrose uptake in onion may be set at some point and remains invariant throughout the day. Similar studies were also conducted on discs cut from mature leaves of Spinacia oleracea var America. Between 1 and 8 hours after the onset of the photoperiod, the sucrose content of the spinach leaves increased from 2.6 to 9.3 micromoles per milligram chlorophyll. A comparison of the kinetic profiles obtained from leaf discs, taken at these times, indicated that sucrose uptake was not influenced by these changes in internal sugar levels. The relationship between the above findings and ;trans' inhibition of exogenous sucrose uptake is discussed. Although intermediate changes in sugar levels in sugar beet leaves did not appear to affect sucrose influx, autoradiographic studies revealed that these changes dramatically affected the partitioning of exogenously supplied [(14)C]sucrose. Our results indicate that while intermediate changes in internal sugar levels have little effect on sucrose influx across the plasmalemma, they may dramatically affect partitioning between the phloem and the mesophyll vacuole.

Journal Article↗