Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current.

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.     

Macrophage catabolism of lipid A is regulated by endotoxin stimulation

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.     

Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos.

The mago nashi (mago) locus is a newly identified strict maternal effect, grandchildless-like, gene in Drosophila melanogaster. In homozygous mutant mago females reared at 17 degrees C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84-98% of the embryos die. In contrast, at 25 degrees C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7-14. Furthermore, embryos collected from mago1 homozygous females contain no apparent functional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phenotype). Thus, we propose that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo.     

PCR-generated conspecific sodium channel gene probe for the house fly.

A segment of the house fly (Musca domestica) homologue of the para (paralytic) sodium channel gene of Drosophila melanogaster was isolated by using mixed sequence oligonucleotide primers in the polymerase chain reaction (PCR). The specificity of the procedure was demonstrated by genomic Southern analysis using the housefly PCR amplification product as a probe and by DNA sequence analysis. The latter showed structural homology to the para gene, but not to the corresponding region of DSC1, another D. melanogaster gene with structural similarity to vertebrate sodium channel genes.     

Gelled diet for screwworm (Diptera: Calliphoridae) mass production

A rearing system based on a diet gelled with Water-Lock G-400, a synthetic superabsorbent (poly(2-propenamide-co-2-propenoic acid, sodium salt)) (WL), was compared with the standard rearing system (liquid diet suspended in acetate fibers) for the mass production of screwworms, Cochliomyia hominivorax (Coquerel). The WL rearing system yielded 2% heavier pupae, 32% higher egg to pupa survival, and required 54% less diet and 88% less labor than the standard rearing system. Other advantages of the WL system include reduced susceptibility to suboptimal environmental conditions and labor practices and characteristics conducive to centralization and mechanization of rearing procedures.     

Interleukin-4 activates ion channels in B lymphocytes.

The lymphokine interleukin-4 (IL-4) has been shown to induce dramatic changes in the physiology of resting B cells. We have applied the patch clamp technique in the cell attached and inside/out configurations to resting and IL-4-treated B cells to determine whether specific ion conductances result as a consequence of IL-4 action. We report here that two distinct ion channel events occur in B lymphocytes after treatment with IL-4, (i) induction of an inward rectifying K+ channel that is not observed in untreated cells, and (ii) activation of a large conductance anion channel that is normally silent in non-treated cells in the cell attached patch configuration. These data present the first evidence of a direct effect by IL-4 on ion channels and we suggest roles for these two ionic conductances in IL-4-induced B cell activation.     

Larvae of Maculinea rebeli, a large-blue butterfly, and their Myrmica host ants: wild adoption and behaviour in ant-nests

The behavioural interactions between caterpillars of Maculinea rebeli Hir. and their Myrmica ant hosts were studied, both in the wild at the time of adoption, and inside captive nests of six Myrmica species.
In the wild, freshly moulted, final instar caterpillars left their food-plants at a time of day that coincided with the peak foraging activity of Myrmica (18:00-20:00 h). Once on the ground, caterpillars made no attempt to search for Myrmica but settled and waited for foraging ants to find them, which took up to 1.5 h. There was no adoption ritual: foragers of any Myrmica species picked up the caterpillars within 1–4 sec of discovery, and carried them directly to their nests.
Caterpillars grew from < 2 mg to 110 mg in laboratory ant-nests. About 60 mg was gained in autumn but 40% of this was lost during the winter, while the temperature was < 14 °C. Although caterpillars survived best with their normal host, Myrmica schencki , they could also survive in the nests of other Myrmica species. The presence of queen ants had no effect upon survival. The behaviour of the caterpillars was described and illustrated: this included the production of secretions that were drunk by the ants, begging for food and direct feeding by ants. The preferred solid food was ant eggs.
The results are discussed in terms of the social biology of Myrmica ants. It is hypothesized that Maculinea rebeli caterpillars mimic the touch pheromones of ant worker-larvae. This would explain the inability of ants to recognize caterpillars before touching them, their immediate adoption by any Myrmica species after discovery, host specificity inside wild ant-nests, the absence of queen-effect and the intimate attention of host workers.