The retinoid X receptor (RXR) participates in a multitude of nuclear receptor signaling pathways and is induced by its highly sought-after cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by the ecdysone receptor that consists of a heterodimer comprising the ecdysone receptor monomer (EcR) and the invertebrate RXR homolog ultraspiracle (USP); the latter, however, is believed to have diverged from its RXR origin. From the more evolutionarily-primitive insect Locusta migratoria (Lm), long and short RXR transcripts (LmRXR-L and LmRXR-S, respectively) were detected during embryogenesis. This thesis reports the immunochemical detection of RXR cross-reactive material throughout Locusta embryogenesis, suggesting that the protein may have another role besides ecdysone signaling. Thus, the RXR isoforms were cloned for recombinant expression and purification in order to demonstrate retinoid specificity. Both isoforms bound 9-cis-RA and all-trans-RA with high affinity. Binding was further corroborated by the identification of endogenous retinoids during embryogenesis. Embryos were first subjected to modified “Bligh and Dyer” as well as solid phase extractions to circumvent oil precipitation that rendered whole homogenates unsuitable for retinoid assay and detection. The RA-inducible Cyp26A1-promoter reporter cell line identified the presence of endogenous RAs (5.4 nM) from insect embryo extracts. Finally, high pressure liquid chromatography followed by mass spectroscopy (HPLC/MS) confirmed the identity of all-trans-RA and the more abundant 9-cis-RA (1.3 nM). These findings suggest a functional role for 9-cis-RA in the invertebrate embryo and favour signaling through the combination of 9-cis-RA and RXR in evolutionarily early RA-driven animal development. / Thesis (Master, Biology) -- Queen's University, 2007-12-13 10:14:43.474

The rectal intima of the desert locust was found to possess fixed negative charges, rather than fixed neutral sites. It was suggested that the molecular species responsible for the negative sites might be acidic amino acids. The selective permeability of the intima as estimated from diffusion potentials, for divalent cations was 3a⁺² > Ca⁺² > Sr⁺² > Mg⁺² > Mn⁺², for monovalent cations was NH₄⁺ > Rb⁺ > Cs⁺ > K⁺ > Na⁺ > Li⁺ > TEA⁺ and for monovalent anions was HCO₃⁻ > CN⁻ > F⁻ > NO₃⁻ > CL⁻ > CH₃COO⁻ > Br⁻ > H₂PO₄⁻ > I⁻. Cation affinity for the fixed charged site was found to be in the order of Ca⁺² > Mg⁺⁺ » K⁺ > Na⁺. Similarity of effects of pH and ion concentration on streaming and diffusion potentials indicated that ion movement and water flow might take place through the same route. The intima was found to act as an osmotic compartment such that at high external osmotic pressures, the rate of water flow was reduced due to a shrinkage of the effective pore size in the intima, however the relative permeability of ions did not seem effected by membrane dehydration. Unstirred layers at the membrane-solution interfaces were found to have a minimal effect on diffusion potentials, however half of the value for streaming potentials was found to be due to a diffusion potential caused by an ion concentration difference in opposing unstirred layers. Calcium -45 flux across the intima at pH 5.5 (i.e. possessing fixed charge) was found to be 81 times greater, at a concentration of 10 mM/l CaCl₂, than calcium flux at the same concentration across the uncharged membrane (pH 2.2). The same effect was not significant for rubidium. Conversely, the removal of fixed charge enhanced anion flux. Calcium permeation rate was found to be a function of its dissociation rate from the fixed charge and did not correlate in a simple manner with the membrane binding capacity for calcium. A trans effect on calcium flux was also found in the intima and is believed to be a function of the dissociation rate of calcium from the fixed negative site. It was concluded that electro-osmosis was not the mode of water movement across the rectum, however physiological advantage of electro-osmosis was discussed. Flux experiments possible indicate that the intima might be the rate limiting step for K⁺ reabsorption in a hydrated animal. / Science, Faculty of / Zoology, Department of / Graduate

An in vitro preparation of the rectum of the desert locust Schistocerca gregaria is described and characterized. The rectal epithelium was mounted as a flat sheet separating two well stirred chambers and the trans-membrane potential difference (PD), membrane resistance, and short circuit current (SCC) were monitored. The PD and membrane resistance remained relatively constant for at least six hours at 35 mV (lumen positive) and 6000 Ohms . cm ⁻², respectively. After an initial two hour transient period during which the SCC dropped from 8 uMoles of charge .cm⁻² .hr⁻¹ to 3.5 uMoles. cm⁻² .h⁻¹ an approximate steady-state condition was reached and maintained for at least another four hours. The SCC was consistant with either a net cation transport from the hemolymph to the lumen of the rectum or a net anion transport in the opposite direction. Since Cl⁻ was the only major anion in the bathing media some experiments were carried out to evaluate the contribution of active Cl⁻ transport to the SCC. Substitution of SO4⁻ of NO3⁻ during the third and fourth hours in vitro had no effect on the SCC, although they abolished the initial two hours transient. Similar experiments in which Cl⁻ was substituted by acetate had a complex stimulatory effect on the SCC. Measurements of ³⁶Cl⁻ fluxes under SCC conditions during the same time period demonstrated a mean net flux of 1.52 uMoles of Cl⁻ cm⁻².hr from the rectal lumen to the hemolymph. This value is equivalent to one half the simultaneous SCC. These experiments clearly demonstrate that the rectal epithelium actively transports Cl⁻ in a direction consistent with the observed SCC but that this active transport of Cl⁻ does not contribute to the SCC during the third and fourth hours in vitro. Since rectal Na+ and K+ transport reported by other workers is in the wrong direction to account for the observed SCC, it seems necessary to propose (1) a Cl⁻ exchange pump (probably with HCO3⁻) perhaps located on the luminal membrane to account for the fact that active Cl⁻ transport does not contribute to the SCC and (2) a H+ and/or HCO3⁻ pump to account for the SCC. These proposals are incorporated in a model for organization of ion transport processes in the locust rectum. / Science, Faculty of / Zoology, Department of / Graduate

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