Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

Fifth (last) instar nymphs of th e tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to th e more polar conjugates API. Only small quantities of free hormone were transferred to th e hemolymph and the carcass within t h e first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [³H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of API conjugates. API obtained in second instar nymphs fed with [3H]ecdysone ([³H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of APT remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., API mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle. Apolar products (mainly AP2) that accumulated in eggs of females injected with [³H]E or [³H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to API, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs. We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.     

Voltage dependence of theChara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide

Summary It is generally agreed that solute transport across theChara plasma membrane is energized by a proton electrochemical gradient maintained by an H⁺-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H⁺-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the trueI-V profile for the pump when treatments alter the thermodynamic pressure on transport.We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cellI-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl₃, so that steady-state currents could be recorded under voltage clamp between –400 and +100 mV. Exposures to 1mm NaCN (CN) and 0.4mm salicylhydroxamic acid (SHAM) depolarized (positive-going)Chara membrane potentials by 44–112 mV with a mean half time of 5.4±0.8 sec (n=13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3±0.9 sec ([ATP] _t=0, 0.74±0.3mm; [ATP] _t=x , 0.23±0.02mm). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses ofdI-V curves (±CN+SHAM) as well as of families ofI-V curves taken at times during CN+SHAM exposures indicated a stoichiometry for the pump of one charge (H⁺) transported per ATP hydrolyzed and an equilibrium potential near –420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60–75% of the total membrane conductance nearV _m. Complementary results were obtained also in fitting previously publishedI-V data gathered over the external pH range 4.5–7.5 Kinetic features deduced for the pump were dominated by a slow step preceding H⁺ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium. These characteristics predict, in marked contrast to the situation forNeurospora, that cytoplasmic acid loads inChara should shift the pumpI-V curve negative-going along the voltage axis with little change in maximum current output at positive voltages.     

Amino acid sequences of stomach and nonstomach lysozymes of ruminants

Summary Complete amino acid sequences are presented for lysozymesc from camel and goat stomachs and compared to sequences of other lysozymesc. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants.     

Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells

Chromaffin cells cultured in serum-free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum-containing medium. Addition of insulin-like growth factor-I (IGF-I) to serum-free medium restored high K(+)-stimulated catecholamine secretion to the levels seen in serum-treated cultures. In contrast, addition of IGF-I to serum-containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF-I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF-I not only enhanced high K(+)-stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl-phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF-I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF-I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2(+)-dependent catecholamine secretion was slightly, but consistently, greater from IGF-I-treated cells than from untreated cells. Our results suggest that IGF-I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.     

Relationship of indole-3-butyric acid and adventitious rooting in M.26 apple (Malus Pumila mill.) shoots cultured in vitro

The role of indole-3-butyric acid (IBA) in adventitious root formation was studied by analyzing the uptake and subsequent metabolism of IBA in shoots of M.26 apple (Malus pumila Mill.) rootstock grown in vitro. Roots were induced by exposing shoots to 4 M IBA and [³H]IBA for 5 days in the dark and then transferring them to plant growth regulator (PGR)-free medium in the light until roots formed. Approximately 50% of the total radioactivity applied was taken up from the agar medium by the shoots during the 5-day incubation period in IBA. Indole-3-butyric acid metabolism was studied by extraction and high-performance liquid chromatographic (HPLC) separation of [³H]IBA and metabolites from the basal sections of treated shoots. The major [³H]IBA metabolite co-eluted with authentic [¹⁴C]indole-3-acetic acid (IAA) suggesting that IBA was converted to IAA in the shoots. The proportion of newly synthesized IAA present as conjugates was higher at the end of the 5-day IBA treatment period than after 13 days in PGR-free medium. There appeared to be no conjugation of IBA at any time.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.