Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms

The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.We are very grateful to Dr. Roger Ellis (SCRI, Dundee, Scotland) for the gift of barley seeds, and to Drs Roger Ellis, Alan Schulman and Cathie Martin for helpful advice and comments during the course of this work.     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.     

Dielectric properties of human blood and erythrocytes at radio frequencies (0.2–10 MHz); dependence on cell volume fraction and medium composition

The dielectric properties of human erythrocytes (red blood cells) suspended in whole blood and in isotonic media at various volume fractions (haematocrits) have been studied in the frequency range 0.2–10 MHz, in which the so-called-dispersion due to the Maxwell-Wagner effect is known to occur. The capacitance and conductance at 25 °C were measured by an instrument interfaced to a computer. The rectangular sample cavity (1 ml volume) contained four pure gold electrode pins, and the sample could be circulated by a roller pump. The frequency-dependence of the permittivity and conductivity were fitted by non-linear least squares regression. Corrections were applied for non-linearity in the dielectric increment at high haematocrit, and for electrode polarisation when diluting the blood in saline. Data were interpreted in terms of a simple equivalent resistor-capacitor circuit. From the measured haematological values the specific membrane capacitance (C_m) and the conductivities internal and external to the cells (_i and _o respectively) were estimated. The conductivities behaved in a predictable manner with a mean of 0.458 S · m^–1 (s.d. ± 0.044) for _i, whereas the value of C_m (and indeed the actual capacitance of the suspension) was dependent on the amount of plasma present. Hence, in stationary normal (anticoagulated) whole blood samples, C_m was as high as 2.98 F · cm^–2 (s.d. ± 0.40), in contrast to about 0.9 F · cm^–2 in blood diluted more than two-fold (to less than 20% hct) in isotonic media. The high value remained when the diluent was plasma. The C_m value returned to a high value when washed erythrocytes were reconstituted with plasma, provided that this was present at above a critical or threshold concentration of about 30 vol % in the medium, irrespective of the haematocrit in the range studied (15–44%). The C_m remained low in serum. When added to washed cells in saline, purified fibrinogen had no effect. However, high C_m values were obtained by fibrinogen supplementation to serum and diluted plasma. Applying moderate flow to whole blood approximately halved its high C_m value in an exponential manner with flow rate, whilst the C_m of washed cells (31–67% hct) slightly increased, and converged to the value for whole blood under flow. We interpret the highapparent C_m value in stationary samples to be a result of rapid cell aggregation in the presence of plasma, where rouleaux formation takes place before visible sedimentation sets in.     

Behavior of Colonial Orb-weaving Spiders during a Solar Eclipse

The behavior of colonial orb-weaving spiders (Metepeira incrassata) in tropical Veracruz, Mexico was studied during the total solar eclipse on July 11, 1991. Spiders behaved in a manner typical of daily activity until totality, when many began taking down webs. After solar reappearance, most spiders that had begun taking down webs rebuilt them. There was no significant difference in the overall activity patterns of spiders during totality across a range of colony sizes. Experimental illumination of part of a colony during totality altered web takedown behavior. While spiders in the darkness of totality began to take down webs, those spiders which were artificially illuminated did not. These observations suggest that the primary environmental cue responsible for the daily rhythm of web building behavior in this species is light level.     

Palisade mesophyll cell expansion during leaf development in Zinnia elegans (Asteraceae)

Temporal and spatial patterns of palisade mesophyll cell expansion in Zinnia elegans were characterized as a basis for developing a suspension culture model for mesophyll cell expansion. Our objectives were to 1) identify the leaf regions from which cells in various stages of expansion could be selectively isolated for culture, and 2) develop a basis for comparison of rate and extent of mesophyll cell expansion in culture with that in the leaf. Palisade mesophyll cells were isolated from expanding leaves by gentle physical maceration without the use of enzymes. Isolated cells from leaves in different stages of expansion were then measured by computer image analysis. Analysis of size frequency distributions showed that unexpanded cells can be isolated from the entire blade of small leaves or the basal regions of partially expanded leaves. Fully expanded cells can be obtained from the apical and middle regions of partially expanded leaves. Within the leaf, Zinnia mesophyll cells expanded from about 400 μm² to about 2.300 μm² at an estimated rate of 160 μm² d^-1. The percent increase in cell length exceeded the percent increase in cell width. Expansion of mesophyll cells continued for 6–8 d after epidermal expansion ceased. This difference in the timing of cell expansion in epidermal and mesophyll cells indicates that different regulatory factors may be operating in these adjacent tissues and underscores the importance of investigating the regulation of mesophyll cell expansion at the cellular level.     

Sympathetic and sensory innervation of the urinary tract in young adult and aged rats: a semi-quantitative histochemical and immunohistochemical study

Summary The sympathetic innervation of the urinary tract of young adult (4 months) and aged (24+ months) rats has been examined by glyoxylic acid-induced fluorescence for the detection of noradrenaline and by immunofluorescence using antisera against tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Immunostaining for calcitonin gene-related peptide (CGRP), known to be present in pelvic sensory nerves, was also performed. Semi-quantitative estimations of nerve densities were made of noradrenergic and peptidergic fibres innervating the smooth musculature of the ureter, bladder and urethra, and of the urinary tract vasculature. In the aged rats the overall patterns of innervation remained unchanged. However, with the exception of the vesical vasculature, the density of noradrenergic innervation decreased as did the intensity of histofluorescence. A similar pattern of results was observed by TH and NPY immunofluorescence. The results present evidence for a diminution in the sympathetic control of the urinary tract in aged rats. The pattern and density of CGRP-immunoreactive nerves was unchanged in the aged animals suggesting that pelvic visceral sensory innervation is more resistant to the effects of advancing age.