Germination of Monocolpate Angiosperm Pollen: Effects of Inhibitory Factors and the Ca2+-Channel Blocker, Nifedipine

Hydration of pollen of Narcissus pseudonarcissus was retardedand germination blocked in media with supra-optimal concentrationsof osmoticum. Activation of the grains, expressed in circulatorymovement in the vegetative cell, was not blocked. Wall developmentwas disrupted, and pectic material and callose were depositedthroughout. In the absence of calcium many grains burst on hydration.The survivors showed evidence of activation, but few tubes wereformed. In medium with supra-optimal Ca²⁺, activation proceeded,but where tube tips were produced they became occluded withcallose, which eventually formed a general lining to the intine.Nifedipine, a Ca²⁺-blocker, did not prevent activation at 10^–4M, but reduced callose deposition and inhibited polarized movementin the vegetative cell. Prominences formed at the germinationsites were mostly low and rounded. During recovery in normalmedium, tube tips with normal callose linings were formed. Colchicine,a microtubule inhibitor, had no effect on activation or germination.Cytochalasin D, an actin inhibitor, prevented activation ofthe vegetative cell, but did not arrest all wall deposition.Movement began soon after transfer to normal medium, and somegrains produced adventitious tube tips. While Ca²⁺ appears notto be essential for activation, these results may be interpretedas indicating links in the normal course of germination betweenthe initial Ca²⁺ influx at the potential germination sites and:(a) polarization of movement in the vegetative cell, probablyrelated to re-orientation of the actin cytoskeleton; and (b)patterned deposition of callose, which appears to have an importantmorphogenetic role. Narcissus pseudonarcissus, pollen activation, pollen germination, osmotic effects, actin cytoskeleton, nifedipine, cytochalasin D, colchicine, role of Ca²⁺ flux     

DNA condensation by an oxidizable cationic detergent. Interactions with lipid vesicles.

Cationic amphiphile-mediated delivery of plasmid DNA is the non-viral gene transfer method most often used. In the present work, we considered a new cysteine-detergent, ornithinyl-cysteinyl-tetradecylamide (C(14)-CO), able to convert itself, via oxidative dimerization, into a cationic cystine-lipid. By using fluorescence techniques, we first characterized the structure of complexes of plasmid DNA with C(14)-CO molecules either kept as monomers, or oxidized into dimers. Both forms are able to condense DNA, with the formation of hydrophobic micelle-like domains along the DNA chain. Domains with a larger molecular order were obtained with dimeric C(14)-CO/DNA complexes. In a second step, the interactions of these complexes with lipid vesicles considered as membrane models were investigated. In the presence of vesicles, we observed a decondensation of the DNA involved in complexes obtained with C(14)-CO monomers. With anionic vesicles, the DNA is released into the bulk solution, while with neutral vesicles, it remains bound to the vesicles via electrostatic interactions with inserted C(14)-CO molecules. In sharp contrast, the complexes with C(14)-CO dimers are unaffected by the addition of either neutral or anionic vesicles and show no interaction with them. These results may partly explain the low transfection efficiency of these complexes at the +/-charge ratios used in this study.     

Characterization of immunomodulatory properties and accessory cell function of small intestinal epithelial cells.

We have studied the immunomodulatory properties of epithelial cells from the small intestine on T cell immune function in vitro. Proliferation of lymph node cells stimulated either with antigen or with mitogen was inhibited by epithelial cells in a dose-dependent fashion. The epithelial cell-mediated suppression of lymphocyte proliferation was blocked by indomethacin, a cyclooxygenase pathway inhibitor, demonstrating that the suppressive effect of epithelial cells was related to prostaglandin secretion. Furthermore, the action of epithelial cell-secreted prostaglandin on lymphocytes was related to its effect on IL-2 as the suppressive effect of epithelial cells was abrogated by the addition of exogenous IL-2. As previously reported, epithelial cells constitutively express MHC class II and we found them able to present antigen in a class II-restricted fashion when their suppressive effects were blocked by indomethacin. Furthermore, epithelial cells activated by LPS secrete an IL-1 like molecule in a fashion analogous to other antigen-presenting cells. These results demonstrate that epithelial cells can both enhance and suppress in vitro T cell immune responses and further characterize the mechanisms by which intestinal epithelial cells may function in gut-associated immune responses.     

Characteristics of the range of mutations in nystatin resistance induced in yeasts by 6-N-hydroxylaminopurine

A simple procedure for induction of nistatin-resistant mutants in yeasts under the action of 6-N-hydroxylaminopurine (HAP) is proposed. Some differences between the spectra of mutations induced by HAP and UV-light are observed: HAP induces dominant and double recessive mutants more frequently.     

Measurement of positional isotope exchange rates in enzyme-catalyzed reactions by fast atom bombardment mass spectrometry: application to argininosuccinate synthetase

Fast atom bombardment mass spectrometry (FAB-MS) has been used to measure positional isotope exchange rates in enzyme-catalyzed reactions. The technique has been applied to the reactions catalyzed by acetyl-CoA synthetase and argininosuccinate synthetase. The FAB technique is also able to quantitatively determine the oxygen-18 or oxygen-17 content of nucleotides on as little as 10 nmol of material with no prior derivatization. Acetyl-CoA synthetase has been shown by FAB-MS to catalyze the positional exchange of an oxygen-18 of ATP from the beta-nonbridge position to the alpha beta-bridge position in the presence of acetate. These results are consistent with acetyl adenylate as a reactive intermediate in this reaction. Argininosuccinate synthetase was shown not to catalyze a positional isotope exchange reaction designed to test for the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase was also found not to catalyze the transfer of oxygen-18 from [ureido-18O]citrulline to the alpha-phosphorus of ATP in the absence of added aspartate. This experiment was designed to test for the transient formation of carbodiimide as a reactive intermediate. These results suggest that either argininosuccinate synthetase does not catalyze the formation of citrulline adenylate or the enzyme is able to completely suppress the rotation of the phosphoryl groups of PPi.