Z-linked inheritance of male olfactory response to sex pheromone components in two species of tortricid moths, Ctenopseustis obliquana and Ctenopseustis sp.

The olfactory response from male pheromone sensitive sensilla was investigated in the endemic New Zealand brownheaded leafrollers Ctenopseustis obliquana (Walker) and C. sp. ropeana (Lepidoptera, Tortricidae). The responses from 281 sensilla from the parental strains and from both the reciprocal crosses, including F₁, F₂ and maternal and paternal backcrosses were recorded, and statistically analysed using a multivariate analysis.In males of both the parental strains, a large amplitude cell responded to the main pheromone component of the conspecific female, in C. obliquana (Z)-8-tetradecenyl acetate (Z8-14:OAc) and in C. sp ropeana (Z)-5-tetradecenyl acetate (Z5-14:OAc). Both male types also possessed a small amplitude cell, which in C. obliquana responded weakly to Z5-14:OAc and tetradecyl acetate (14:OAc), and in C. sp ropeana responded to Z8-14:OAc. The responses from the different types of hybrid males were more variable than the responses from parental males. A main pattern could, however be seen, corresponding with the expected pattern in a sex-linked inheritance on the Z-chromosome of a C. sp ropeana type dominant genetic factor. The more pronounced variation in the hybrids could not be explained by this model, and might be due to the involvement of additional genes.

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Résumé Les réactions olfactives des sensilles mâles sensibles aux phéromones ont été examinées par enregistrement de l'extrémité de la sensille chez les tordeuses C. obliquana Walker et C. sp. ropeana. Les enregistrements ont porté sur 281 sensilles des lignées parentales et des croisements réciproques de F₁, F₂ et de croisements en retour maternel et paternel. Les résultats des enregistrements d'une sensille ont été soumis à une analyse en composantes principales.Chez les mâles de chaque lignée parentale un seul type physiologique de sensille a été découvert; une cellule répond par un pic grand au principal constituant de la phéromone femelle conspécifique. (Z)-8-acétate tétradécényl (Z8-14:OAc) pour C. obliquana, et (Z)-5-acétate tétradécényl (Z5-14:OAc) pour C. sp. ropeana. Une seconde type de cellule dans les sensilles des deux espèces de mâles présente un pic petit pour Z5-14:OAc et pour l'acétate tétradécyl (14:OAc) chez C. obliquana, et pour C. sp. ropeana au Z8-14:OAc. Les réponses des sensilles des différents types de mâles hybrides sont plus hétérogènes que celles des sensilles de leurs pères. Un schéma général pourrait cependant être décelé, correspondant au schéma prévu avec une hérédité d'un facteur dominant liée au sexe sur le chromosome Z de C. sp. ropeana. La variation plus accentuée chez les hybrides ne peut être expliquée par ce modèle, et pourrait impliquer des gènes additionnels.

     

Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes

Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio-Beads SM-2, the functional interaction between receptor and adenylate cyclase was restored. Adenylate cyclase was stimulated by hCG, HCG+GTP or GppNHp and NaF. Reconstituted proteoliposomes bound more ¹²⁵I-hCG (528 fmol/mg protein) than membrane-bound receptors (384 fmol/mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Regulation of nicotinic acetylcholine receptor function by adenine nucleotides

1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.     

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Selective labeling of α-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions

The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C. S., Manshouri, T., and Atassi, M. Z. (1987)J. Prot. Chem. 6, 455–461; Garcia-Borron, J. C., Bieber, A. L., and Martinez-Carrion, M. (1987)Biochemistry 26, 4295–4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.     

Acetylcholinesterase activity in antennal receptor neurons of the sphinx moth Manduca sexta

Summary We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the antenna of the sphinx moth Manduca sexta. High levels of echothiophate-insensitive (presumably intracellular) AChE activity were found in six different types of antennal receptors localized in specific regions of the three antennal segments of the adult moth. Mechanosensory organs in the scape and pedicel, the Böhm bristles and Johnston's organ, are innervated by AChE-positive neurons. In each annulus of the antennal flagellum, AChE-positive neurons are associated with six sensilla chaetica and a peg organ, probably a sensillum styloconicum. At least 112 receptor neurons (8–10 per annulus) innervating the intersegmental membranes between the 14 distalmost annuli also exhibit high levels of echothiophate-resistant AChE. In addition, each annulus has more than 30 AChE-positive somata in the epidermis of the scale-covered (back) side of the flagellum, and 4 AChE-positive somata reside within the first annulus of the flagellum. Since none of the olfactory receptor neurons show a high level of echothiophateresistant AChE activity, and all known mechanoreceptors are AChE-positive, apparently intracellular AChE activity in the antenna correlates well with mechanosensory functions and is consistent with the idea that these cells employ acetylcholine as a neurotransmitter.     

Local anesthetic action of phentolamine on insect mechanoreceptors

1. The effect of phentolamine on the response properties of insect mechanoreceptors and on the conduction in their axons was examined using electrophysiological techniques. 2. Phentolamine blocked conduction of action potentials along axons, an effect which exhibited 3 characteristics typical of local anesthetics: the effect was frequency-dependent, reversible and varied for nerves with different diameters. 3. The concentration of phentolamine required to block axonal conduction (1-2 x 10(-3) M) was significantly higher than that required to abolish the response of receptors to mechanical stimulation (3-5 x 10(-4) M). 4. All mechanoreceptors that were examined in Locusta migratoria and Periplaneta americana were inactivated by phentolamine (Table 1). The type I receptors (chordotonal, campaniform and hair sensilla) were inactivated within 5-15 min following phentolamine application. The only type II receptor examined (forewing stretch-receptor) underwent a phase of repetitive discharge before being inactivated. 5. Tolazoline and metoclopramide inactivated, like phentolamine, mechanoreceptors at lower concentrations than necessary to block axonal conduction. However, yohimbine and chlorpromazine inactivated mechanoreceptors and blocked axonal conduction at similar concentrations. 6. These findings suggest that phentolamine affects sense-organ specific ionic processes that are more sensitive to the drug than the ionic processes along the axons.