Formation of apolar ecdysteroid conjugates by ovaries of the house cricket Acheta domesticus in vitro.

The newly laid eggs of the house cricket Acheta domesticus contain apolar ecdysteroid conjugates, which we have hypothesized to be ecdysone long-chain fatty acyl esters [Whiting & Dinan (1988) J. Insect Physiol., in the press]. The ovaries of mature adult female A. domesticus in vitro convert [3H]ecdysone into apolar conjugates identical with those found in newly laid eggs. Comparison of the radioactive metabolites produced on incubation of [3H]ecdysone with various organs of adult female A. domesticus in vitro indicate that the fat-body is the major producer of polar ecdysteroid metabolites at this stage of development, whereas the ovaries are the major site of production of apolar metabolites. Apolar metabolites are also produced to a lesser extent by the crop, gut sections and the fat-body. Hydrolysis of radioactive metabolites produced by the ovaries with Helix enzymes releases only [3H]ecdysone, and thus ecdysone is not metabolized before conjugation by the ovaries. Formation of chemical derivatives (acetonide and acetates) of these 3H-labelled apolar conjugates strongly indicates that the position of conjugation is through the hydroxy group at C-22 of ecdysone. Extensive chromatographic analysis of the 3H-labelled apolar metabolites produced by the ovaries by t.l.c. and h.p.l.c. and comparison with authenticated reference compounds have conclusively demonstrated that the conjugates consist of ecdysone esterified at C-22 to a mixture of common long-chain fatty acids. The major fatty acyl esters have been identified and their percentage contribution to the mixture determined: laurate (0.5%), myristate (2.8%), palmitate (25.8%), stearate (8.4%), arachidate (1.0%), oleate (15.7%), linoleate (38.8%) and linolenate (2.1%). In addition there are three minor unidentified peaks, one of which has been tentatively identified as ecdysone 22-palmitoleate (2.6%). Comparison of this percentage composition with the previously published fatty acid composition of A. domesticus haemolymph [Wang & Patton (1969) J. Insect Physiol. 15, 851-860] reveals remarkable similarities, indicating that the acyl transferase(s) forming the conjugates have a broad specificity with regard to the fatty acyl substrate.     

Islet-cell Carcinoma (Zollinger—Ellison Syndrome) with Fulminating Adrenocortical Hyperfunction and Hypokalemia

The production of ACTH-like material by tumours arising in non-endocrine tissue may initiate severe adrenocortical hyperfunction. The pathogenesis and clinical and laboratory features of Cushing''s syndrome associated with such tumours are characteristic. The autonomous production by the tumour of ACTH-like material cannot be suppressed by exogenous corticoids. The onset of clinical symptoms is rapid; muscle wasting, general weakness, thirst and peripheral edema predominate, and the classical signs of Cushing''s syndrome may be absent. High levels of plasma 17-hydroxycorticosteroids and urinary 17-hydroxycorticosteroids and 17-ketosteroids, usually with normal levels of urinary aldosterone, commonly occur. Hypokalemic alkalosis unresponsive to replacement therapy may cause death. In the case reported herein, the intriguing possibility exists that two hormone-like substances were produced by the primary growth and its metastases: one, ACTH-like, to account for the adrenal hyperplasia and Cushing''s syndrome; and another, gastrin-like, giving rise to the ulcerogenic diathesis.     

Certain withanolides from Iochroma gesnerioides antagonize ecdysteroid action in a Drosophila melanogaster cell line

Sixteen withanolides isolated from Iochroma gesnerioides (Kunth) Miers (Solanaceae) have been assessed for their activities as ecdysteroid agonists and antagonists. None of the compounds showed any agonistic activity, but several showed significant antagonistic activity. With a 20-hydroxyecdysone concentration of 5 × 10^–8 M, the ED₅₀ values for 2,3-dihydro-3-methoxywithaferin A, 2,3-dihydro-3-methoxywithacnistine, 2,3-dihydro-3-methoxyiochromolide and 2,3-dihydro-3-hydroxywithacnistine are 3.5 × 10^-5 M, 1 × 10^–5 M, 5 × 10^–6 M and 2.5 × 10^–6 M, respectively.     

GABAB Receptor-Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice

Abstract: Previously, we have shown a significant increase in number of GABA_B receptor binding sites in neocortex and thalamus of lethargic ( lh/lh ) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABA_B receptors would inhibit [³H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age-matched male lh/lh and +/+ mice were similar in uptake of [³H]GABA. In the neocortical preparation, baclofen dose-dependently inhibited [³H]GABA release evoked by 12 m M KCl, an effect mediated by GABA_B receptors. The maximal inhibition ( I _max) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC₅₀ (3 µ M ) was unchanged. In the thalamic preparation, the effect of baclofen (50 µ M ) was 58% less robust in lh/lh mice. Other effects mediated by GABA_B receptors (inhibitions in Ca²⁺ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABA_B receptor-mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABA_B receptors on GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.     

A defective conditioned behavior and a defective adenylate cyclase in theDrosophila mutantrutabaga

Summary TheDrosophila X-linked mutantrutabaga (Duerr and Quinn 1982) fails to perform normally in olfactory conditioning paradigms, in spite of being able to sense odorants and shock (Figs. 1–3).rut is capable of forming an association between shock and odorant, but memory decays rapidly; the memory of the mutant following intensive training resembles that of normal flies following very brief training (Fig. 4).rut flies also display in vitro a defective adenylate cyclase activity (Fig. 6). The enzyme in the mutant is responsive to stimulation by a putative neurotransmitter and by a guanyl nucleotide (Fig. 8) but the activity is lower than normal even in the presence of forskolin (Fig. 8) and MnATP (Fig. 9), suggesting that the lesion is closely associated with the function of the catalytic subunit.rut/ + heterozygotes are semi-recessive with regard to both the behavioral defect and the biochemical defect (Figs. 5, 7). The behavioral and the biochemical lesions detected inrut flies are discussed in light of current molecular models of learning.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.