ALBERT

Notes: Abstract Confocal fluorescence microscopy enables visualisation and quantitation of fluorescent probes at high resolution deep within intact tissues, with minimal disturbance both of cell–cell interactions and the mechanical, ionic and physiological effects of the extracellular matrix. We illustrate the principles of multiple-parameter 3-D (x,y,z) imaging using reconstruction of nuclear channels in mammalian cells. Repeated sampling in time generates 4-D (x,y,z,t) images which can be used to follow dynamic changes, such as blue-light-dependent chloroplast re-orientation, in intact tissues. Quantitative measurements from multi-dimensional images require calibration of the spatial dimensions of the image and the fluorescence intensity response. This must be determined throughout the volume, which must be sampled to correct for geometric distortion as well as photometric errors arising from the complete optical system, including the specimen. The effects of specimen calibration are illustrated for morphological analysis of stomatal closing responses to abscisic acid in Commelina from 4-D images. Calibrated 4-D imaging allows direct volume measurements and we have followed volume regulation of chondrocytes in cartilage explants during osmotic perturbation. In intact cartilage, unlike in isolated cells, the chondrocytes exhibit volume regulatory mechanisms. In other cases, the fluorescence intensity of the probe may be related to a physiological parameter of interest and changes in its distribution within the cell. Optical sectioning permits discrimination of signal in separate compartments within the cell and can be used to follow transport events between different organelles. We illustrate 3-D (x,y,t) measurements of vacuolar glutathione conjugate pump activity in intact roots of Arabidopsis by following the sequestration of a fluorescent conjugate between glutathione and monochlorobimane. Dynamic measurements of protein localisation are now possible following the introduction of chimeric fusion proteins with green fluorescent protein (GFP) from Aequoria victoria. We have analysed the disposition of heterochromatin in nuclei of living Schizosaccharomyces pombe cells expressing a chimeric construct between Swi6 and GFP. Heterochromatin dynamics can be followed throughout mitosis in 4-D (x,y,z,t) images. Statistical analysis of the fluorescence histograms from each nucleus over time provides quantitative support for aggregation and dispersion of Swi6-GFP clusters during mitosis, rather than dissociation of Swi6 from the heterochromatin. A wide range of single-wavelength and ratio probes are available for imaging different ion activities. We compare 3-D (x,y,t) measurements of ion activities made using single-wavelength (Fluo-3 for calcium) and ratio (BCECF for pH) measurements, using stomatal responses in Vicia faba to peptides from the auxin-binding protein of maize and tip growth in pollen tubes of Lilium longiflorum as examples. Ratioing techniques have many advantages for quantitative fluorescence measurements and we conclude with a discussion of techniques to develop ratioing of single-wavelength probes against alternative references, such as DNA, protein or cell wall material.

Notes: Abstract   The cellulase gene cel3 has been isolated from Agaricus bisporus and sequenced. The 5′-end of the cel3 transcript was determined by primer extension and S1 nuclease protection. Putative regulatory elements have been identified in the cel3 promoter and 3′-untranslated regions. The cel3 coding region is interrupted by six short introns, two of which separate the coding regions for the three modules in the CEL3 protein: cellulose-binding domain, linker region, and catalytic domain. Three of the remaining four introns are positioned in regions coding for loops between structural moieties. Intron positions are conserved between cel3 and other related cellulases.

Notes: Summary The phenoxy herbicides MCP and 2,4,5-T undergo extensive conjugation with plant constituents. ThereFore, in order to measure total herbicide residues, a method must be used which will extract and hydrolyze such conjugates. The studies reported here showed that heating plant tissue under dilute alkaline conditions is adequate to measure total herbicide residues. Using the alkaline extraction method, it was shown that there were no detectable residues of MCP or 2,4,5-T in the grain of wheat plants treated with these herbicides.

Notes: The fourth-order dispersion relation for D(3He) obtained from a straightforward small k⊥ρD expansion does not properly reproduce the exact kinetic dispersion relation near ion–ion hybrid resonance. It is shown that, in general, whenever the majority is not resonant the expanded dispersion relation may break down. A method to obtain a correct approximate fourth-order dispersion relation has been developed.

Notes: The power reflection coefficient for D(H), DT(3He), and D (3He) ion cyclotron heating is found in closed form. The fast wave equation previously solved numerically is solved by a slowly varying amplitude expansion about the Budden equation asymptotics. The coupling between the fast Alfvén wave and the ion Bernstein wave is described by this formalism. The dissipation associated with the ion cyclotron resonance is accounted for by the slow variation. Validity criteria for this method are presented.