Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.

The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.     

Theory of the locomotion of nematodes: Dynamics of undulatory progression on a surface

We develop a model of the undulatory locomotion of nematodes, in particular that of Caenorhabditis elegans, based on mechanics. The model takes into account the most important forces acting on a moving worm and allows the computer simulation of a creeping nematode. These forces are produced by the interior pressure in the liquid-filled body cavity, the elasticity of the cuticle, the excitation of certain sets of muscles and the friction between the body and its support.

We propose that muscle excitation patterns can be generated by stretch receptor control. By solving numerically the equations of motion of the model of the nematode, we demonstrate that these muscle excitation patterns are suitable for the propulsion of the animal.

     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Regulation of passive potassium transport of normal and transformed 3T3 mouse cell cultures by external calcium concentration and temperature

Summary Regulation of passive potassium ion transport by the external calcium concentration and temperature was studied on cell cultures of 3T3 mouse cells and their DNA-virus transformed derivatives. Upon lowering of external calcium concentration, passive potassium efflux generally exhibits a sharp increase at about 0.1mm. The fraction of calcium-regulated potassium efflux is largely independent of temperature in the cases of the transformed cells, but shows a sharp increase for 3T3 cells upon increasing temperature above 32°C. In the same range of temperature, the 3T3 cells exhibit the phenomenon of high-temperature inactivation of the residual potassium efflux at 1mm external calcium. At comparable cellular growth densities, the transformed cell lines do not show high-temperature inactivation of residual potassium efflux. These results are consistent with the notion of a decisive role of the internal K⁺ concentration in the cell-density dependent regulation of cell proliferation. In particular, the growth-inhibiting effect of lowering the external Ca²⁺ concentrations is considered as largely due to a rise of passive K⁺ efflux and a subsequent decrease of internal K⁺ concentration. The experimental data on the Ca²⁺ dependence of passive K⁺ flux are quantitatively described by a theoretical model based on the constant field relations including negative surface charges on the external face of the membrane, which cooperatively bind Ca²⁺ ions and may concomitantly undergo a lateral redistribution. The present evidence is consistent with acidic phospholipids as representing these negative surface charges.This work is dedicated to the memory of Max Delbrück (deceased March 10, 1981), in whose laboratory in 1966 the earlier version of the present theoretical model was developed by one of the authors.