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M R Knight

Publications and source records attributed to M R Knight.

At least 37 records · Page 2Linked to original sources
PubMed1996

The moss, Physcomitrella patens, transformed with apoaequorin cDNA responds to cold shock, mechanical perturbation and pH with transient increases in cytoplasmic calcium.

The gene for apoaequorin has been used previously to indicate cytosolic calcium changes in higher plants. Here we report the transformation of the moss Physcomitrella patens with the cDNA for apoaequorin. Stable transformants were obtained in the wild type which reconstitute the calcium-sensitive luminescent protein aequorin in vivo after incubation in coelenterazine, and continue to grow normally. The wild type responds to cold-shock (0-10 degrees C) with increases in cytosolic calcium. Mechanical perturbation, in the form of touch, also induces transient increases in cytosolic calcium. A smaller response to pH, distinct from the touch response and exhibiting different kinetics, can also be detected.

Aequorin↗
PubMed1996

Calcium imaging shows differential sensitivity to cooling and communication in luminous transgenic plants.

Imaging of a recombinant bioluminescent Ca2+ indicator, aequorin, in an entire organism showed three novel features of Ca2+ signals in plants. First, cooling the plant from 25 degrees C to 2 degrees C demonstrated differential sensitivities between organs, the roots firing a Ca2+ signal at some 8-10 degrees C higher than the cotyledons. Secondly, prolonged cooling provoked Ca2+ oscillations, but only in the cotyledons. These oscillations occurred with a frequency of 100 s and damped down within 800 s. Thirdly, cooling the roots of mature plants triggered a Ca2+ signal in the leaves, as a result of organ-organ communication. However, warming and then recooling the roots did not generate a second Ca2+ signal in these leaves. This desensitisation was not due to down-regulation in the leaf since this was able to generate a Ca2+ signal of its own when cooled directly. Thus a combination of a recombinant bioluminescent indicator with photon counting imaging reveals startling new aspects of signalling in intact organs and whole organisms.

Aequorin↗
PubMed1996

Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation.

Cold shock elicits an immediate rise in cytosolic free calcium concentration ([Ca2+]cyt) in both chilling-resistant Arabidopsis and chilling-sensitive tobacco (Nicotiana plumbaginifolia). In Arabidopsis, lanthanum or EGTA caused a partial inhibition of both cold shock [Ca2+]cyt elevation and cold-dependent kin1 gene expression. This suggested that calcium influx plays a major role in the cold shock [Ca2+]cyt response and that an intracellular calcium source also might be involved. To investigate whether the vacuole (the major intracellular calcium store in plants) is involved, we targeted the calcium-dependent photoprotein aequorin to the cytosolic face of the vacuolar membrane. Cold shock calcium kinetics in this microdomain were consistent with a cold-induced vacuolar release of calcium. Treatment with neomycin or lithium, which interferes with phosphoinositide cycling, resulted in cold shock [Ca2+]cyt kinetics consistent with the involvement of inositol trisphosphate and inositide phosphate signaling in this response. We also investigated the effects of repeated and prolonged low temperature on cold shock [Ca2+]cyt. Differences were observed between the responses of Arabidopsis and N. plum-baginifolia to repeated cold stimulation. Acclimation of Arabidopsis by pretreatment with cold or hydrogen peroxide caused a modified calcium signature to subsequent cold shock. This suggests that acclimation involves modification of plant calcium signaling to provide a "cold memory."

Acclimatization↗
PubMed1995

Calcium and the generation of plant form.

The involvement of mechanical signals (tension and compression) in the determination of the form of living organisms has been speculated upon for many years. These mechanical signals (both environmental and those generated within the plant itself) have significant effects on plant development and thus morphology. Plants respond to externally applied mechanical signals (touch and wind) by an immediate elevation of cytosolic calcium concentration ([Ca2+]eyt) in stimulated cells. This response requires the movement of plant tissues to cause tension and compression. Some of the more longer-term responses to mechanical signals, e.g. TCH gene expression and reduction in hypocotyl growth, show a calcium-dependency. It seems likely, therefore, that the effects of mechanical signals on plant development are mediated by the second messenger, calcium. This raises the exciting possibility that this simple ion plays a central role in the determination of plant form itself.

Calcium↗
PubMed1995

Circadian oscillations of cytosolic and chloroplastic free calcium in plants.

Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.

Aequorin↗
PubMed1995

Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin.

Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli. However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established. In order to monitor internal free Ca2+ in E. coli during challenge with chemotactic agents, the Ca(2+)-activated photoprotein aequorin was expressed in a chemotactic strain (AB1157) and a non-chemotactic strain [BL21(DE3)] of E. coli. Repellents were found to cause an increase (50-150 nM) in intracellular free Ca2+, whereas attractants caused a small but consistent decrease in intracellular free Ca2+. These data are in agreement with the proposed model that an increase in intracellular free Ca2+ causes tumbling. The effect of increasing external Ca2+ on the regulation of intracellular free Ca2+ in both strains was monitored by using aequorin. The resting level of free Ca2+ in E. coli (AB1157) was found to be 100 nM, which agrees with previous data [Gangola and Rosen (1987) J. Biol. Chem. 262, 12570-12574]. As these results also show differences in the regulation of intracellular free Ca2+ between the two strains in the presence of high external Ca2+ concentrations, this may have implications for the effect of high-Ca2+ environments on E. coli.

Aequorin↗
PubMed1993

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins.

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.

Aequorin↗
PubMed1992

Wind-induced plant motion immediately increases cytosolic calcium.

Wind is one of the most unusual and more dramatic of the environmental signals to modify plant development. Wind-stimulated crops are also known to experience considerable reductions in growth and subsequent yield. There is at present no experimental data to suggest how wind signals are perceived and transduced by plant cells. We have genetically transformed Nicotiana plumbaginifolia to express aequorin and thus produced luminous plants that directly report cytosolic calcium by emitting blue light. With these plants we have found wind stimulation to cause immediate increases in cytosolic calcium and our evidence, based on the use of specific inhibitors, suggests that this calcium is mobilized from organelle sources. Our data further suggest that wind-induced movement of tissues, by mechanically stimulating and stressing constituent plant cells, is responsible for the immediate elevation of cytosolic calcium; increases occur only when the plant tissue is actually in motion. Repeated wind stimulation renders the cells refractory to further calcium signaling but responsiveness is rapidly recovered when stimulation is subsequently diminished. Our data suggest that mechanoperception in plant cells may possibly be transduced through intracellular calcium. Since mechanoperception and transduction are considered crucial to plant morphogenesis, our observations suggest that calcium could be central in the control and generation of plant form.

Aequorin↗
PubMed1992

Genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.: nucleotide sequence of cDNA clones and initial studies of expression.

The small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the French bean Phaseolus vulgaris L. is encoded by a small gene family consisting of a minimum of three members. Three small subunit genes (rbcS genes) represented in a light-grown primary leaf cDNA library were characterised by sequencing two cDNAs which were full-length and one which was deficient in part of the sequence encoding the transit peptide. The cDNA clones are identical in their coding sequences, for both the transit peptide and the mature polypeptide, but divergent in their untranslated sequences. The derived amino acid sequence is very similar to that reported for other species, although the first amino acid of the mature polypeptide is isoleucine, which differs from the methionine found in all other higher plant rbcS genes. Surprisingly, one of the cDNA clones contains two introns, which are at positions conserved in rbcS genes from other species. It is concluded that this cDNA resulted from the cloning of an unprocessed transcript. Alternative polyadenylation sites are found for two of the genes. Expression of the rbcS genes in the primary leaves is stimulated by light, although transcripts can readily be detected in dark-grown leaves. Expression is also organ-specific, as in other species. The frequency of cDNA clones in the library indicates that the different genes show quantitative differences in expression and S1 nuclease analysis suggests that individual rbcS genes are photoregulated.

Amino Acid Sequence↗
PubMed1991

Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium.

Methods for measuring plant cytoplasmic calcium using microelectrodes or microinjected fluorescent dyes are associated with extensive technical problems, so measurements have been limited to single or small groups of cells in tissue strips or protoplasts. Aequorin is a calcium-sensitive luminescent protein from the coelenterate Aequorea victoria (A. forskalea) which is formed from apoaequorin, a polypeptide of relative molecular mass approximately 22,000, and coelenterazine, a hydrophobic luminophore. Microinjected aequorin has been widely used for intracellular calcium measurement in animal cells, but its use in plants has been limited to exceptionally large cells. We show here that aequorin can be reconstituted in transformed plants and that it reports calcium changes induced by touch, cold-shock and fungal elicitors. Reconstituted aequorin is cytoplasmic and nonperturbing; measurements can be made on whole plants and a calcium indicator can be constituted in every viable cell. Now that apoaequorin can be targeted to specific organelles, cells and tissues, with the range of coelenterazines with differing calcium sensitivities and properties available, this new method could be valuable for determining the role of calcium in intracellular signalling processes in plants.

Aequorin↗
PubMed1991

Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli.

We describe a novel and simple method for the measurement of bacterial cytosolic free calcium ([Ca2+]i) using recombinant aequorin reconstituted within live bacterial cells. Using this method we have measured the effects of external calcium, complement, phagocytosis and antibiotics on the [Ca2+]i of Escherichia coli. In principle this method should be applicable to any genetically transformable organism and should suffer fewer problems than fluorescent dyes for subcellular calcium measurement.

Aequorin↗
PubMed1988

Hypertensive encephalopathy. Magnetic resonance imaging demonstration of reversible cortical and white matter lesions.

Computed tomographic (CT) and magnetic resonance imaging (MRI) views of the brain were obtained in two adults and one child with hypertensive encephalopathy (HTE). Noncontrast CT was normal in one case and demonstrated decreased density posteriorly in two cases; MRI demonstrated focal, symmetric increased signal intensity in white matter and cortex, with occipital lobe involvement in each case. These lesions were better visualized on T2-weighted than on spin density images and were resolved on follow-up MRI four to five weeks later. These MRI studies support the concept that HTE is caused by the multifocal extravasation of fluid and proteins across the blood-brain barrier during "breakthrough" of cerebral autoregulation. We found that MRI appears more sensitive than CT and better defines the anatomy of cerebral involvement in HTE.

Adult↗
PubMed1986

Aortic pseudoaneurysm due to ingested foreign body.

We have described a child with pseudoaneurysm of the thoracic aorta caused by an ingested safety pin, who survived this life-threatening injury in which the diagnosis was elusive for four months. Esophageal foreign bodies, especially sharp, pointed ones, can cause such a cardiovascular complication. A high index of suspicion is imperative if the diagnosis is to be made early. Serial chest x-ray films showing an enlarging mediastinal mass should trigger aggressive radiologic evaluation.

Aorta, Thoracic↗
PubMed1984

The management of clotted angiographic catheters.

In our experience, clotted angiographic catheters pulled back to near the puncture site and severed near the skin will spontaneously clear, facilitating insertion of a guidewire and catheter exchange without repuncture or other manipulation.

Angiography↗