An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Effect of the antioxidant dibunol on the function of the adrenal cortex, the thyroid and the adenohypophysis in adult and old rats

The effect of a single antioxidant (dibunol-D) injection (100 mg/kg body weight) on the functional activity of adrenal cortex (AC), thyroid gland (TG) and tropic hormone production by adenohypophysis (AH) has been studied in old and adult rats. For 48 hours following D administration two-phase changes in adrenocorticotropic function of AH and steroidogenesis were detected in the AC: activation during the first hours was followed by suppression 24 hours later, and recovery 48 hours later. Thyrotropic AH function and thyroidogenesis were found to be decreased during the first hours of D effect. Thyroidogenesis recovery by the end of the first day was delayed as compared to the recovery of AH thyrotropic function. It is suggested that the mechanisms of D action are based on its effects mediated by changes in the functional activity of endocrine glands and associated with resetting of endocrine regulation of body functions.     

The spectral sensitivity of eye movements in response to light flashes inDaphnia magna

Summary The crustaceanDaphnia magna responds to a flash of light with a ventral rotation of its compound eye; this behavior is termed eye flick. We determined the spectral sensitivity for the threshold of eye flick in response to light flashes having three different spatial characteristics: (1) full-field, extending 180° from dorsal to ventral in the animal's field of view; (2) dorsal, 30° wide and located in the dorsal region of the visual field; (3) ventral, same as dorsal but located ventrally. All three stimuli extended 30° to the right and to the left of the animal's midplane. We found that spectral sensitivity varies with the spatial characteristics of the stimulus. For full-field illumination, the relative sensitivity was maximal at 527 nm and between 365 nm and 400 nm, with a significant local minimum at 420 nm. For the dorsal stimulus, the relative sensitivity was greatest at 400 nm, but also showed local maxima at 440 nm and 517 nm. For the ventral stimulus, the relative sensitivity maxima occurred at the same wavelengths as those for the full-field stimulus. At wavelengths of 570 nm and longer, the responses to both dorsal and ventral stimuli showed lower relative sensitivity than the full-field stimulus. No circadian or other periodic changes in threshold spectral sensitivity were observed under our experimental conditions. Animals which had their nauplius eyes removed by means of laser microsurgery had the same spectral sensitivity to full-field illumination as normal animals. Our results are discussed in terms of our current knowledge of the spectral classes of photoreceptors found in theDaphnia compound eye.