Modulation of asialoglycoprotein binding activity in livers of pregnant or estrogen-treated rats

This report deals with the modulation of activity and expression of the hepatic asialoglycoprotein receptor, in pregnant or diethylstilbestrol-treated rats.The results show a two-fold increase in the total cell associated binding activity, both in pregnant and in estrogen-treated animals, with respect to normal values. On the contrary the surface expression was shown to be strongly enhanced only in the liver of pregnant rat. Therefore the modulation shown by this receptor system in pregnancy seems to be only partially estrogen-dependent.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

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