Characterization of a class of small auxin-inducible soybean polyadenylated RNAs

Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 M 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.     

In vitro organogenesis and plant regeneration from leaves of Solanum candidum Lindl,S. quitoense Lam. (naranjilla) and S. sessiliflorum Dunal

Adventitious shoots and roots were regenerated from leaf segments of 3 Solanum species: S. candidum Lindl., S. quitoense Lam. and S. sessiliflorum Dunal. Leaf explants differentiated shoots on modified MS medium supplemented with 23–163 M kinetin and 0–5.7 µM indoleacetic acid (IAA). Excised shoots were induced to form roots by transfer to media with benzyladenine (BA) and naphthaleneacetic acid (NAA) at 0.09 and 0.11 µM respectively for S. quitoense and 0.01 µM NAA for S. candidum and S. sessiliflorum. Adventitious roots were produced directly from leaf explants with 0–140 µM kinetin and 0–5.7 µM IAA in combination. Rooted plants were successfully established in the greenhouse.     

Design and application of a versatile triple-laser cell and chromosome sorter

A high-resolution triple-laser sorter was designed and constructed to provide flexible switchover and high-resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual-laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual-laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual-laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red-labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple-laser system.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased incidence of abnormal foetuses in the offspring of cyclophosphamide-treated male mice

The incidence of morphologically abnormal foetuses in the litters of cyclophosphamide (CP)-treated male mice was investigated and compared with control values. In two experiments (100 mg/kg) CP was shown to increase the incidence of grossly abnormal foetuses over that seen in the controls, although neither was statistically significant in isolation. When the probabilities from the two tests of significance were combined using the method of Fisher the result was significant (P = 0.02). These results suggest that an acute exposure to a mutagen in male mice can cause genetic damage that results in an increased incidence of phenotypically abnormal offspring. However, the large numbers of animals required and the variable control level of abnormalities, indicate that this dosing regimen is an inefficient method of studying the genetic mechanisms responsible for the effects seen.     

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for -L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 m diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.     

Structural determinants of cationic amphiphilic amines which induce clear cytoplasmic vacuoles in cultured cells

Disobutamide (D), an antiarrhythmic cationic amphiphilic amine (CAA), was withdrawn from clinical testing when clear cytoplasmic vacuoles (CCV) were found in the rat and dog during toxicity studies. To delineate the structural determinants of amines that induce CCV, we exposed cultured rat urinary bladder carcinoma and rabbit aorta muscle cells to numerous cationic drugs and chemicals and examined cells by phase light microscopy. The cationic moiety of these CAA was responsible for the induction of CCV. The very potent inducers were compounds that had two strongly basic amine (cationic) centers. The bis tertiary amines were particularly potent inducers. Aliphatic diamines of minimal lipophilicity-induced CCV, thus showing that an "amphiphilic" structural feature, though present in many CAA drugs, is not necessary for CCV induction. The distance between the two cationic centers was irrelevant to the induction of CCV. These results support the concept that CCV are a manifestation of intracellular (e.g., intralysosomal) drug storage. These structural delineations will be useful in future drug design and for further understanding of drug-cell interactions. Based on these findings, we were able to synthesize an antiarrhythmic CAA which did not induce CCV.     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Light inactivation of glucose 6-phosphate dehydrogenase is rapid and occurs before photosynthetic O₂ evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced change in the kinetic parameters of glucose 6-phosphate dehydrogenase is in maximal velocity.