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Biomedical subjects

D B Morton

Publications and source records attributed to D B Morton.

At least 19 recordsLinked to original sources
PubMed1997

Up- and downregulation of esr20, an ecdysteroid-regulated gene expressed in the tracheae of Manduca sexta.

Investigations of ecdysteroid-regulated gene cascades in Drosophila have shown that characteristics of downstream genes in such cascades include their repression by high ecdysteroid levels, their expression at low hormone levels, and the dependence of their expression on protein synthesis. In an earlier study, we identified a gene, esr20, which is expressed in the tracheae of the tobacco hornworm, Manduca sexta, prior to larval and pupal ecdyses. Initial characterization of the expression of esr20 suggested that it had the above characteristics of a downstream gene in an ecdysteroid-regulated cascade. The present study shows that, unlike the downstream genes in Drosophila, the expression of esr20 in tracheae cannot be induced by changes in the ecdysteroid levels alone. We present evidence which suggests that a decline in ecdysteroid is necessary but not sufficient for expression. Soon after pupal ecdysis the level of the esr20 transcript drops fourfold, and by 24 h after ecdysis the transcript is undetectable. Evidence is presented which suggests that this decline in transcript levels requires protein synthesis and appears to result from a decline in the stability of the transcript.

Animals↗
PubMed1996

Insect physiology: the emerging story of ecdysis.

The discovery of a new insect peptide hormone that triggers ecdysis - shedding of an old cuticle - has revealed hidden layers of intricacy about an insect behavior previously thought to be mediated by a single neuropeptide.

Animals↗
PubMed1996

Neuropeptide-stimulated cyclic guanosine monophosphate immunoreactivity in the neurosecretory terminals of a neurohemal organ.

The neuropeptide eclosion hormone acts on the nervous system of the tobacco hornworm, Manduca sexta, to increase cyclic guanosine monophosphate (cGMP) levels. In this study I describe the localization of some of the sites where these increases occur. Prior to pupal ecdysis, eclosion hormone stimulates an increase in cGMP in a network of fibers in the transverse nerve of each abdominal ganglion. Double-label experiments with propidium iodide suggest that the cGMP immunoreactivity is primarily localized in neurosecretory nerve endings. The time course of the increase in cGMP immunoreactivity and its requirement for lipid metabolism is similar to that of the cGMP increase measured by radioimmunoassay. The cGMP response in the transverse nerve is stage-specific, occurring prior to pupal ecdysis and not prior to larval or adult ecdysis.

Animals↗
PubMed1996

Expression of a developmentally regulated gene, Mng10, in identified neurosecretory cells in the CNS of Manduca sexta.

We are interested in the molecular events underlying the development of the nervous system of Manduca sexta during the final 24 h of the pupal molt. In this article we describe a gene, Mng10, that is expressed in the abdominal nervous system of M. sexta and is developmentally regulated over this 24-h period. In situ hybridization analysis shows that the transcript is localized predominantly to a single pair of uniquely identifiable neurosecretory neurons, the NS-L1 cells in the abdominal ganglia. Mng10 is a single copy gene encoding a 229 amino acid protein with a predicted molecular mass of 26 kDa. At the amino acid level the protein shows 34% identity to the yeast transcription unit, Yer082. Northern blot analysis shows that the transcript of Mng10 is very rare, comprising about 0.001% of the poly (A)+ RNA from the CNS and is detectable at 4 h but not 24 h prior to pupal ecdysis. One of the physiological events that develops over the final 24 h of the pupal molt is the ability of the nervous system to respond to the neuropeptide eclosion hormone. In this context, it is interesting to note that the NS-L1 cells are members of the group of 50 neurons that show increased cGMP immunoreactivity when the nervous system is exposed to the neuropeptide eclosion hormone.

Amino Acid Sequence↗
PubMed1996

Identification of a developmentally regulated gene, esr16, in the tracheal epithelium of Manduca sexta, with homology to a protein from human epididymis.

Events related to both ecdysis and metamorphosis require the expression of a new set of genes, the majority of which are regulated by the changes in ecdysteroid levels. We have initiated studies to identify genes whose expression is up-regulated between 24 and 4 h before pupal ecdysis in Manduca sexta. In this paper we report the partial characterization of one such gene, esr16. The transcript of esr16 is detected by Northern blot analysis in nervous tissue, muscle and trachea isolated from animals 4 h before, but not 24 h before pupal ecdysis. In situ hybridization showed that the transcript was expressed in epithelial cells of the large tracheae surrounding the nervous system and muscle. Sequence analysis suggested that the gene encoded a secreted protein with 35% identity to HE1, a human epididymal-specific gene.

Amino Acid Sequence↗
PubMed1996

Comparison of the expression patterns of five developmentally regulated genes in Manduca sexta and their regulation by 20-hydroxyecdysone in vitro.

A previous study, using subtractive hybridization, identified five genes (esr16, esr20, Mng10, Mng14 and tps9) whose transcripts were up-regulated prior to metamorphosis in Manduca sexta nervous tissue. The developmental time points chosen for subtraction suggested that expression could be coordinately regulated and should be negatively regulated by the steroid hormones, the ecdysteroids. In the present paper, we present an analysis of the expression patterns of these five genes, using reverse transcription-polymerase chain reaction (RT-PCR), at various times during development and assess the effects of 20-hydroxyecdysone and cycloheximide on their expression in vitro. This analysis revealed that with the exception of esr20 all transcripts were detectable at all times and that all but one of the genes were up-regulated in vivo and in vitro in the absence or in the presence of low levels of the ecdysteroids. In the absence of ecdysteroids, cycloheximide blocked the accumulation of only two transcripts, esr16 and esr20. These results reveal a much more complex pattern of gene expression in the central nervous system prior to metamorphosis than previously imagined.

Animals↗
PubMed1996

Subtractive hybridization strategy using paramagnetic oligo(dT) beads and PCR.

Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. The subtracted cDNA is then amplified with PCR and used for library screening. Using this method, we have identified four cDNA clones that represent developmentally regulated transcripts in the central nervous system of the tobacco hornworm Manduca sexta. All four transcripts are of low abundance, comprising only 0.001%-0.5% of the poly(A)+ RNA pool.

Animals↗
PubMed1995

Eclosion hormone-stimulated cGMP levels in the central nervous system of Manduca sexta: inhibition by lipid metabolism blockers, increase in inositol(1,4,5)trisphosphate and further evidence against the involvement of nitric oxide.

Previous studies have shown that the neuropeptide, eclosion hormone, stimulates a nitric oxide-independent increase in the levels of cGMP in the nervous system of Manduca sexta. By contrast, recent results in Bombyx mori suggest that eclosion hormone increases cGMP via the production of nitric oxide. In view of these conflicting results we have carried out additional studies to test whether nitric oxide is involved in this process in Manduca. Evidence presented here supports our earlier observations that in Manduca the eclosion hormone-stimulated increase in cGMP is nitric oxide- and carbon monoxide-independent. In addition, we show that a wide variety of inhibitors of lipid metabolism block the eclosion hormone-stimulated cGMP increase. This supports the hypothesis that the activation of the guanylate cyclase is mediated by a lipid messenger. We also show that eclosion hormone stimulates an increase in the levels of inositol(1,4,5)trisphosphate. The time-course of this increase is consistent with the hypothesis that eclosion hormone stimulation of a phospholipase C is an early event in the cascade that results in an increase in cGMP. Receptor-mediated lipid hydrolysis is often mediated by G protein-coupled receptors. Experiments using pertussis toxin show that the eclosion hormone-stimulated increase in cGMP is not mediated by a pertussis toxin-sensitive G protein.

Animals↗
PubMed1995

Effects of acute and chronic restraint on the adrenal gland weight and serum corticosterone concentration of mice and their faecal output of oocysts after infection with Eimeria apionodes.

Experiments were designed to investigate the potential stress of procedures commonly used for restraining mice and to discover whether habituation of the adrenocortical response occurred when chronic restraint was used. The study also examined the effect of chronic restraint on the faecal output of oocysts by mice trickle infected with Eimeria apionodes. The results showed that restraining mice for one hour was stressful and that restraining them repeatedly for one hour daily for seven or 21 days did not lead to habituation of the adrenocortical response. Restraint for one hour daily for seven days did not cause the recurrence of a clinical infection in mice which were chronically infected with E apionodes. Restraining mice for one hour daily for seven days before and for 14 days during the infection also had no effect on the prepatency, patency or intensity of an E apionodes infection.

Adrenal Glands↗
PubMed1995

Remodeling of the insect nervous system.

Our nervous systems and behavior are shaped by hormonally driven developmental changes that continue beyond the embryonic period. Key insights into this process have emerged from studies of the insect nervous system. During insect metamorphosis, the nervous system is remodeled through postembryonic neurogenesis, programmed cell death and the modification of persistent neurons. These changes are regulated to a large degree by gene cascades that are triggered by steroid hormones, the ecdysteroids. Current studies are attempting to reveal the molecular mechanisms involved in regulating these dramatic examples of developmental plasticity.

Animals↗
PubMed1995

Stress measurements in mice after transportation.

Experiments were performed using physiological measures and behavioural parameters to find the acclimatization period in mice to common scientific procedures. Corticosterone levels were significantly elevated in mice killed immediately after being moved to an experimental room (P < 0.05) but levels returned to the normal in less than 1 day, despite mice being exposed to additional stressors such as novel environment, new cages, new bedding material, separation from their cage mates, regrouping, isolation in individually housed mice and a new handler. Behaviours such as rearing, climbing, grooming, feeding and sexual, changed significantly immediately after transportation of mice but most of these behaviours stabilized relatively quickly. In spite of the corticosterone levels, our behavioural observations suggest that even 4 days were not enough to allow the mice to acclimatize fully.

Acclimatization↗
PubMed1995

Corticosterone, adrenal and spleen weight in mice after tail bleeding, and its effect on nearby animals.

Experiments were performed in mice to study if stress was involved in tail bleeding and to investigate any transmittable signal at killing. A second study looked at the time taken to recover from stress elicited by these procedures. Corticosterone levels were significantly higher in mice immediately after the completion of tail bleeding than in control mice (P < 0.05) suggesting that tail bleeding in mice was stressful. This study did not show any evidence for an odour or sound being released during killing or tail bleeding as there was no significant effect on corticosterone levels in mice present in the same room at the time these procedures were carried out. Corticosterone levels in mice killed on days 1, 3, and 7 after tail bleeding were significantly lower (P < 0.05) than the average corticosterone level in tail blood on day 1, indicating that mice recovered within 24 h from the stress of tail bleeding.

Adrenal Glands↗
PubMed1995

Effect of cycloheximide on eclosion hormone sensitivity and the developmental appearance of the eclosion hormone and cGMP regulated phosphoproteins in the CNS of the tobacco hornworm, Manduca sexta.

The neuropeptide, eclosion hormone (EH), triggers ecdysis behavior at the end of each molt in Manduca sexta. Previous studies have shown that the action of EH is mediated by an increase in cGMP and is associated with the phosphorylation of two proteins, named the EGPs. The ability of insects to respond to EH is developmentally regulated with sensitivity being first seen at about 8 hr prior to the normal time of ecdysis. The EGPs are also first detectable in the CNS at 8 hr prior to ecdysis, suggesting that it is their synthesis which determines EH sensitivity. The protein synthesis inhibitor, cycloheximide was used to study the development of the events leading to pupal ecdysis in Manduca. The results of these experiments suggest that protein synthesis is necessary about 10 hr before ecdysis for both the development of EH sensitivity and for the appearance of the EGPs.

Animals↗