Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.     

The ectoparasitic wasp Eulophus pennicornis (Hymenoptera: Eulophidae) uses instar-specific endocrine disruption strategies to suppress the development of its host Lacanobia oleracea (Lepidoptera: Noctuidae)

To successfully complete its development, the gregarious ectoparasitoid Eulophus pennicornis must inhibit the moult of its host, Lacanobia oleracea. In the present study, we examined the possibility that moult- and metamorphosis-associated endocrine events may be disrupted in caterpillars parasitized as newly moulted last (sixth) instars. Juvenile hormone (JH) titres on days 2 and 5 of the final stadium were significantly higher (> 100 fold) in parasitized than in non-parasitized hosts, in which JH was essentially absent. Elevated JH levels were associated with reduced haemolymph JH esterase (JHE) activity (down by 99.8%) and enhanced in vitro JH biosynthesis by the corpora allata (CA) (up to 4.5 fold). Wasp adults and/or larvae, in which we measured high levels of JH III (up to 2.7 ng/g), but little or no JH I or JH II, were not seen as likely sources of JH in parasitized hosts, in which we found mostly JH I and JH II. In addition, removal of parasitoid eggs or larvae after oviposition did not prevent the rise in JH titres seen in parasitoid-laden hosts, suggesting that wasp venom may be responsible for the observed hormonal dysfunction. Host haemolymph 20-hydroxyecdysone (20-E) levels were largely unaffected by parasitism during the final stadium although they were observed to increase earlier and decrease more rapidly in parasitized insects. We compare these results with those reported earlier for L. oleracea larvae parasitized by E. pennicornis as penultimate (fifth) instars, which display significantly depressed 20-E titres relative to control larvae. We conclude that E. pennicornis employs host endocrine-disruption strategies that differ according to whether the host is parasitized as a penultimate or final-stadium larva.     

Fast induction of vitellogenin gene expression by juvenile hormone III in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae).

The present paper describes the effect of juvenile hormone III (JH III) upon vitellogenin (Vg) gene expression in cardioallatectomized females of Blattella germanica. Northern blot analyses of time course studies showed that Vg mRNA can be detected 2 h after the treatment with 1 microgram of JH III. Western blot analyses revealed that Vg protein is detectable 4 h after the same treatment. The study of the influence of the age showed that 48-h-old females seem more sensitive than 24-h-old females, whereas differences were less apparent between 48- and 72-h-old females. Dose-response studies indicated that 0.01 microgram of JH III is ineffective, whereas the doses of 0.1, 1 and 10 micrograms induced the synthesis of Vg in a dose-dependent fashion. Finally, the administration of three successive doses, of 0.01 microgram of JH III each, did not result in detectable Vg production, whereas two doses of 0.01 microgram followed by one of 1 microgram of JH III induced a greater response than that resulting from a sole dose of 1 microgram of JH III, which suggests that sub-effective doses of JH III elicit a priming effect on Vg production.     

Cimetidine and ranitidine: comparison of effects on hepatic drug metabolism.

Paired studies of hepatic microsomal function were conducted in eight subjects during treatment with two histamine H2 antagonists, cimetidine and ranitidine. Cimetidine but not ranitidine inhibited the metabolism of antipyrine (phenazone) and demethylation of aminopyrine (aminophenazone) as measured by breath 14CO2 production after intravenous injection of 14C-aminopyrine. These results suggest that the metabolic inhibitory actions on the liver may be separated from H2 antagonist effects, and that ranitidine has an advantage over cimetidine by not inhibiting microsomal drug oxidative function.     

Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes

Calcitonin gene-related peptide (CGRP) exerts a positive contractile response directly in rat ventricular cardiomyocytes. This response is mediated by receptors of the CGRP₁-subtype. Amylin is 46% homologous with CGRP and binds to receptors selective for CGRP in a range of tissues. The ability of amylin to influence ventricular contractility has been assessed using cardiomyocytes isolated from the ventricles of adult rats. Cardiomyocytes were subjected to biphasic electrical stimulation at 0.5 Hz. CGRP produced a concentration-dependent positive contractile response which became maximal 4 min after initial stimulation. CGRP increased the contractile amplitude maximally at 1 nM and to a value which was 23.3% greater than in the absence of peptide (EC₅₀ VALUE = 21 pM). Amylin increased the contractile amplitude maximally at 20 nM and to a value which was 17.3% greater than in the absence of peptide (EC₅₀ VALUE = 216 pM). In the presence of amylin (20 nM), the concentration-dependence of the contractile response to CGRP was shifted to the left, so that the response became maximal when CGRP was present at 50 pM. In the presence of CGRP_8–37 (100 nM), a selective antagonist at CGRP₁-preferring receptors, the concentration-dependence of the contractile response to CGRP was shifted to the right (dose RATIO = 54). Similarly, in the presence of CGRP_8–37 (100 nM), the contractile response to amylin was inhibited significantly (P ≤ 0.01). Amylin_8–37 (100 nM) did not inhibit the concentration-dependence of the contractile responses to CGRP and amylin significantly (dose RATIOS = 4.2 and 2.4, respectively). In conclusion, these data indicate that amylin exerts a contractile response directly in rat ventricular cardiomyocytes via CGRP₁-preferring receptors. This effect could assume greater significance in non-insulin-dependent diabetes mellitus and in hypertensive states, in which the concentration of amylin is elevated in plasma.     

A YAC contig across the fragile X site defines the region of fragility.

The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.     

A central role for hepatocyte growth factor in adipose tissue angiogenesis

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor produced in human adipose tissue. In this study, we use 3T3-F442A preadipocytes to study the contribution of HGF to angiogenesis in an in vivo fat pad development model. As observed for human adipocytes, HGF is synthesized and secreted by 3T3-F442A preadipocytes and mature adipocytes. HGF knockdown with small-interfering RNA reduced HGF mRNA expression 82.3 +/- 4.2% and protein secretion 82.9 +/- 1.4% from 3T3-F442A preadipocytes. Silencing of HGF resulted in a 70.5 +/- 19.0% reduction in endothelial progenitor cell migration to 3T3-F442A-conditioned medium in vitro. 3T3-F442A preadipocytes injected under the skin of mice form a fat pad containing mature, lipid-filled adipocytes and a functional vasculature. At 72 h postinjection, expression of the endothelial cell genes TIE-1 and platelet endothelial cell adhesion molecule (PECAM)-1 was decreased 94.4 +/- 2.2 and 91.5 +/- 2.5%, respectively, in 3T3-F442A fat pads with HGF silencing. Knockdown of HGF had no effect on differentiation of 3T3-F442A preadipocytes to mature adipocytes in vitro or in vivo. In developing fat pads under the skin of HGF overexpressing transgenic mice, TIE-1 and PECAM-1 mRNA was increased 16.5- and 21.4-fold, respectively, at 72 h postinjection. The increase in gene expression correlated with immunohistochemical evidence of endothelial cell migration in the developing fat pad. These data suggest that HGF has a central role in regulating angiogenesis in adipose tissue.     

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.