Metabolism of ecdysteroids in the female tickAmblyomma hebraeum (Ixodoidea,Ixodidae): accumulation of free ecdysone and 20-hydroxyecdysone in the eggs

Summary [³H]-20-hydroxyecdysone ([³H]-20E) injected intoAmblyomma hebraeum females 7 days before the beginning of oviposition,viz. at the beginning of vitellogenesis, was converted to 3 polar peaks of unknown nature called 1, 2 and 3, and to apolar conjugates AP1, AP2 and AP3. AP2 have the same retention times as the esters of 20E with long chain fatty acids described inOrnithodoros moubata (Diehl et al. 1985). However, principally unmetabolized 20E was incorporated into the ovaries, and 16% of the injected labelling was recovered in the eggs, 3/4 being free 20E. When 20E was injected during oviposition, it was not converted to the polar products but only to the apolar products. At this time, 76% of the total radiolabel injected accumulated in the egg-batch, principally in the form of free unmetabolized 20E.After injection of [³H]-ecdysone (³H]-E), the three polar metabolites 1, 2 and 3, probably 20-deoxy homologues of 1, 2 and 3 described above were always produced irrespective of the time of injection. In addition, E was metabolized to 20E and to the apolar conjugates AP1, AP2, and AP3. E, 20E and peak 2 were incorporated into the ovary within the first day after injection. These 3 compounds were found in freshly laid eggs in variable proportions, the quantity of E decreasing with time while 20E and peak 2 increased. At the end of oviposition, ca. 60% of the injected radiolabel had been incorporated into the eggs. Apolar products and polar metabolites accumulating in the body were apparently not used as a source of free hormone for the eggs.Our results with tritiated ecdysteroids confirm our data concerning endogenous ecdysteroids of the eggs ofA. hebraeum (Connat et al. 1985). This species, in contrast to 2 other female ticks,Ornithodoros moubata andBoophilus microplus, incorporates free E and 20E instead of ecdysteroid conjugates into its eggs. The role of these free ecdysteroids remains to be elucidated.Abbreviations ES ecdysteroids - E ecdysone - 20E 20-hydroxyecdysone - HPLC high-performance liquid chromatography - RP-18 reyerse phase with C18 aliphatic chains grafted on the silica - RIA radio-immunoassay Part of this work was realized for the PhD thesis of E.M. Dotson     

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.     

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 6. 4-substituted 3-pyridinylalkanoic acids.

(3-Pyridinyl)alkanoic acids substituted at the 4-position with an (arylsulfonamido)alkyl group were synthesized and found to behave as platelet thromboxane receptor antagonists (TxRAs) and thromboxane synthase inhibitors (TxSIs). The compounds behaved as agonists at the vascular receptor for thromboxane A₂.     

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.     

Kinetic studies of lysine-sensitive aspartate kinase purified from maize suspension cultures

Steady state substrate kinetics and feedback regulation properties were determined for lysine-sensitive aspartate kinase (AK) purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures. Two AK isoforms (AK Early and AK Late) were separated by two passages through an anion exchange column as the final steps in a procedure giving 1200-fold purification. Kinetic properties were determined for the major AK Late eluting isoform. Assays were conducted at the pH activity maximum (8.0) and with excess Mg²⁺ to favor a two-substrate reaction involving aspartate and complexed MgATP. AK catalyzed a sequential reaction in which MgATP and aspartate both bind to the enzyme complex before the ADP and aspartyl-phosphate products are released. The K_m value calculated for MgATP was 0.43 millimolar and for aspartate was 1.04 millimolar. Cooperativity in substrate binding was not observed and was not induced by lysine. The lysine concentration required for 50% inhibition of AK activity was 7 micromolar. An apparent Hill coefficient of 1.4 indicated a minimum of two lysine-binding sites on the active AK complex. At nonsaturating substrate concentrations, lysine inhibition was characteristic of an S-parabolic, I-parabolic noncompetitive allosteric inhibitor. The parabolic inhibitor replot, Hill coefficients > 1, and the lack of substrate cooperativity were consistent with a model for multiple lysine-binding sites per active AK subunit. Similar kinetic properties were observed for the AK Early isoform.     

Localization of insulin-like immunoreactivity in the synganglion of nymphal and adult Dermacentor variabilis (Acari: Ixodidae)

Immunocytochemical staining based on a peroxidase-antiperoxidase method showed neurosecretory cells (NSC) reactive to bovine insulin in five of 18 paraldehyde fuchsin-positive neurosecretory regions (NSR) in the synganglion of unfed adult Dermacentor variabilis. This is the first report of a neuropeptide in an ixodid tick. The insulin-specific immunoreactive cells included the posterior medial group of the protocerebral center, posterior group of dorsal opisthosomal center, anterior lateral group of the dorso-lateral cheliceral center, dorsal group of the frontal stomodeal center, and anterior group of the ventral palpal center. After feeding and mating, females no longer had immunoreactive cells in three of five NSR found in virgin, unfed females. However, two cells of the posterior group in dorsal opisthosomal center and anterior lateral group of the dorso-lateral cheliceral center remained immunoreactive throughout feeding. Fed, mated males continued to display immunoreactive cells in four of five NSR found in the virgin, unfed males. All developmental stages of nymphs examined had insulin-specitic immunoreactive cells in two of the five NSR found in unfed adults, including two positively stained cells of the posterior group in dorsal opisthosomal center and anterior group of ventral palpal neurosecretory center.     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.