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Biomedical subjects

G J Allen

Publications and source records attributed to G J Allen.

At least 19 recordsLinked to original sources
PubMed2001

Combining genetics and cell biology to crack the code of plant cell calcium signaling.

Plant hormones, light receptors, pathogens, and abiotic signals trigger elevations in the cytosolic calcium concentration, which mediate physiological and developmental responses. Recent studies are reviewed here that reveal how specific genetic mutations impair or modify stimulus-induced calcium elevations in plant cells. These studies provide genetic evidence for the importance of calcium as a second messenger in plant signal transduction. A fundamental question arises: How can different stimuli use the same second messenger, calcium, to mediate different responses? Recent research and models are reviewed that suggest that several important mechanisms contribute to specificity in calcium signaling in plant cells. These mechanisms include (i) activation of different calcium channels in the plasma membrane and organellar membranes, (ii) stimulus-specific calcium oscillation parameters, (iii) cell type-specific responses, and (iv) intracellular localization of calcium gradients and calcium elevations in plant cells.

Arabidopsis↗
PubMed2001

Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump.

Transgenic plants overexpressing the vacuolar H(+)-pyrophosphatase are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains. These transgenic plants accumulate more Na(+) and K(+) in their leaf tissue than the wild type. Moreover, direct measurements on isolated vacuolar membrane vesicles derived from the AVP1 transgenic plants and from wild type demonstrate that the vesicles from the transgenic plants have enhanced cation uptake. The phenotypes of the AVP1 transgenic plants suggest that increasing the vacuolar proton gradient results in increased solute accumulation and water retention. Presumably, sequestration of cations in the vacuole reduces their toxic effects. Genetically engineered drought- and salt-tolerant plants could provide an avenue to the reclamation of farmlands lost to agriculture because of salinity and a lack of rainfall.

Abscisic Acid↗
PubMed2001

A defined range of guard cell calcium oscillation parameters encodes stomatal movements.

Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores and [Ca2+]cyt oscillations are a fundamental component of stomatal closure. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a 'calcium clamp' and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term 'calcium-reactive' closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was 'calcium programmed' by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.

Abscisic Acid↗
PubMed2001

Guard cell abscisic acid signalling and engineering drought hardiness in plants.

Guard cells are located in the epidermis of plant leaves, and in pairs surround stomatal pores. These control both the influx of CO2 as a raw material for photosynthesis and water loss from plants through transpiration to the atmosphere. Guard cells have become a highly developed system for dissecting early signal transduction mechanisms in plants. In response to drought, plants synthesize the hormone abscisic acid, which triggers closing of stomata, thus reducing water loss. Recently, central regulators of guard cell abscisic acid signalling have been discovered. The molecular understanding of the guard cell signal transduction network opens possibilities for engineering stomatal responses to control CO2 intake and plant water loss.

Abscisic Acid↗
PubMed2001

A statewide assessment of psychosocial rehabilitation programs: general characteristics and services.

Psychosocial rehabilitation (PSR) encompasses a diverse array of skill-building and environmental support services for individuals with serious mental illness. The current study gleaned information from state Department of Mental Heath (DMH) databases and interviews with program directors to describe general characteristics and services of 22 out of 24 PSR programs in one New England state. Results underscored the relative predominance of services targeting social rehabilitation rather than vocational, educational, or residential rehabilitation. In addition, density of service provision across agencies was unrelated to enrollment or daily attendance statistics. Furthermore, low validity coefficients between mandated reporting statistics and information collected via interviews were found, suggesting the need to develop more valid and reliable reporting systems. Finally, four areas of concern were highlighted: substantial discrepancies between average membership enrollment and daily attendance, low percentages of peer-managed activities, predominance of services in traditional clinical settings, and lack of attention to multicultural issues.

Humans↗
PubMed2000

Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant.

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.

Abscisic Acid↗
PubMed2000

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

Abscisic Acid↗
PubMed1999

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↗
PubMed1999

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells.

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

Amino Acid Sequence↗
PubMed1999

Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells.

Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt).

Abscisic Acid↗
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

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells.

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to -100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of approximately 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

Journal Article↗
PubMed1997

Physiological and psychological effects associated with high carbon dioxide levels in healthy men.

BACKGROUND: Perception of CO2 in ambient air may be of fundamental importance to the health, safety, and job performance of persons occupationally exposed to increased levels of CO2. Few studies have examined the perceptual responses to inhaled CO2 at levels between 6-10%. HYPOTHESIS: We hypothesized that healthy, highly-active men would be able to determine the difference between 6% and 8% CO2 concentrations. METHODS: Thirty-two male students (21 +/- 1 yr) served as subjects. Experimental trials (counterbalanced design) included breathing air (control, 21% O2, 79% N2), 6% CO2 (21% O2, 73% N2), and 8% CO2 (21% O2, 71% N2) conditions. The Body Sensations Questionnaire (BSQ) was completed twice during each trial. RESULTS: End tidal CO2 (FETCO2) and BSQ values increased (p < 0.05) as a function of the percentage of inhaled CO2. Respiratory rates during the 8% trial were greater (p < 0.05) than control and 6% CO2 trial measures. BSQ scores were significantly correlated with FETCO2 and respiratory rate measures during the 6% and 8% CO2 conditions. CONCLUSIONS: We conclude that these subjects recognized their exposure to both the 6% and 8% CO2 concentrations, and their responses were more profound during the 8% CO2 condition.

Adult↗
PubMed1996

Construction of a mouse blastocyst cDNA library by PCR amplification from total RNA.

Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT-PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains beta-actin, intracisternal A-type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones.

Animals↗
PubMed1996

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium.

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At 'zero' to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K(+)-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- permeability was assumed then the apparent PCa was lower. However, substitution of Cl- by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl- permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg(2+)-activated and were demonstrated to be Mg(2+)-permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.

Biological Transport↗
PubMed1995

Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose.

Calcium mobilization from intracellular pools couples many stimuli to responses in plant cells. Cyclic adenosine 5'-diphosphoribose (cADPR), which interacts with a ryanodine receptor in certain animal cells, was shown to elicit calcium release at the vacuolar membrane of beet storage root. The vacuolar calcium release pathway showed similarities to cADPR-gated calcium release in animal cells, including inhibition by ruthenium red, ryanodine activation, and high affinity for cADPR [Michaelis constant (Km) = 24 +/- 7 nanomolar]. Analysis by patch-clamping demonstrated that the cADPR-gated pathway in beet is voltage-dependent over the physiological range, does not spontaneously desensitize, and is colocalized with an inositol 1,4,5-trisphosphate (InsP3)-gated calcium release pathway in individual vacuoles.

Adenosine Diphosphate Ribose↗
PubMed1995

Seeing and knowing: knowledge attribution versus stimulus control in adult humans (Homo sapiens).

Interest in cognition in nonhuman animals has inspired new approaches to discovering animals' ability to attribute knowledge to others (e.g., D. J. Povinelli, K. E. Nelson, & S. T. Boysen, 1990). The assumptions of such experiments were tested in this study by training a group of humans (Homo sapiens) to use accurate information provided by a confederate who was watching as 1 container among 4 was baited; a 2nd group was similarly trained to use accurate information provided by a confederate whose back was turned during baiting. On a single reversal trial, the roles of the 2 confederates were switched. Subjects were able to learn their respective tasks but attended to different aspects of the confederates, as revealed by the reversal trial. Although attributional interpretations can be applied to such data, many of the choices in this experiment can be explained more readily with the basic principles of contingency-based learning.

Adolescent↗