Triacylglycerol estolides, a new class of mammalian lipids, in the paracloacal gland of the brushtail possum (Trichosurus vulpecula).

The paracloacal glands are the most prevalent scent glands in marsupials, and previous investigation of their secretions in the brushtail possum (Trichosurus vulpecula) has identified many odorous compounds together with large amounts of neutral lipids. We have examined the lipids by LC-MS, generating ammonium adducts of acylglycerols by electrospray ionisation. Chromatograms showed a complex mixture of coeluting acylglycerols, with m/z from about 404 to 1048. Plots of single [M + NH4](+) ions showed three groups of lipids clearly separated by retention time. MS-MS enabled triacylglycerols and diacylglycerol ethers to be identified from neutral losses and formation of diacylglycerols and other product ions. The earliest-eluting lipids were found to be triacylglycerol estolides, in which a fourth fatty acid forms an ester link with a hydroxy fatty acid attached to the glycerol chain. This is the first report of triacylglycerol estolides in animals. They form a complex mixture with the triacylglycerols and diacylglycerol ethers of lipids with short- and long-chain fatty acids with varying degrees of unsaturation. This complexity suggests a functional role, possibly in social communication.

Arginine-tagging of polymeric nanoparticles via histidine to improve cellular uptake.

Polyarginine, a cell-penetrating peptide, has been shown to aid cellular penetration of bioactives into cells. We utilized a novel approach of using a histidine linker to produce poly(ethyl-cyanoacrylate) (PECA) nanoparticles tagged with oligoarginine and investigated cellular uptake. MALDI TOF/TOF (tandem) analysis revealed that di-arginine-histidine (RRH) covalently bound to PECA nanoparticles to form cationic particles (+18 mV), while longer oligoarginine peptides did not co-polymerize with PECA nanoparticles. Although RRH-tagged nanoparticles had similar size and FITC-dextran entrapment efficiency compared to unmodified nanoparticles, RRH-tagging of nanoparticles resulted in a greater release of FITC-dextran. As the nanoparticles were found to aggregate in Hanks Balanced Salt Solution (HBSS), the effect of phosphate on the zeta-potential of nanoparticles was studied. Treating the nanoparticles with poloxamer-407 prevented aggregation. RRH-tagged PECA nanoparticles increased cellular uptake by a further 30% compared to unmodified PECA nanoparticles and was concentration dependent. We suggest that enhanced cell uptake can be achieved using a di-arginine-histidine construct as opposed to the previously published findings that a minimum of hexa-arginine is necessary. Further, the cationic zeta-potential of the cell-penetrating peptide may not be needed to enhance uptake.

Seasonal changes in RFamide-related peptide-3 neurons in the hypothalamus of a seasonally breeding marsupial species, the brushtail possum (Trichosurus vulpecula).

RFamide-related peptide-3 (RFRP-3) neurons have been shown to inhibit gonadotropin-releasing hormone (GnRH) neuronal activity and hence reproduction in birds and eutherian mammals. They have also been proposed to have a direct hypophysiotropic effect on pituitary gonadotropin release. We used a new RFRP-3 antibody to characterize the cell body distribution and fiber projections of RFRP-3 neurons in the adult female brushtail possum brain. RFRP-3-immunoreactive cell bodies were found scattered within the dorsomedial hypothalamus and the dorsomedial half of the ventromedial hypothalamus, while GnRH neurons were observed scattered rostrocaudally along the lateral septum, rostral to the medial septum. There was a significant 2-fold increase in the RFRP-3 cell body number during the nonbreeding season (summer) compared to the breeding season (winter). Immunoreactive RFRP-3 fibers were distributed throughout the thalamus, preoptic area, and hypothalamus. Very few fibers were observed in the median eminence, especially in the external zone. Intraperitoneal injection of the retrograde tracer Fluoro-Gold resulted in the labeling of 40% of hypophysiotropic tuberoinfundibular dopaminergic (tyrosine hydroxylase-positive) neurons; however, <10% of zona incerta dopaminergic neurons (which are not hypophysiotropic) or RFRP-3 neurons were labeled with this tracer. These observations suggest that RFRP-3 exhibits a seasonal fluctuation in cell numbers, as seen in sheep and birds, which is consistent with an increased inhibitory tone during the nonbreeding season. The lack of RFRP-3 fibers in the median eminence and of Fluoro-Gold uptake from the periphery imply that the actions of this peptide occur primarily centrally rather than at the anterior pituitary gland.

Release and bioactivity of PACA nanoparticles containing D-Lys6-GnRH for brushtail possum fertility control.

Poly(ethylcyanoacrylate) (PECA) nanoparticles containing the chemical sterilitant D-Lys6-GnRH were prepared by an in situ interfacial polymerization technique. Their potential as a peroral delivery system for biocontrol of the brushtail possum, a major pest species in New Zealand, was evaluated. Peptide release from resulting particles was studied in vitro in artificial gastric juice (AGJ), simulated intestinal fluids (SIF) and brushtail possum plasma. The nanoparticles released a small fraction of bioactive over 6h in AGJ and SIF (<5%), while staying intact and retaining fractions of intact D-Lys6-GnRH. In contrast, 60% of D-Lys6-GnRH was released after 1h in possum plasma. The nanoparticles were also administered in vivo into the caecum of brushtail possums. A significant biological response, measured as an increase in plasma luteinizing hormone (LH), was evident 10 min after administration. This demonstrates not only that PECA nanoparticles were able to facilitate the uptake of D-Lys6-GnRH from the caecum into systemic circulation but also that sufficient bioactive peptide reached the pituitary to exert a significant LH response following GnRH receptor mediated endocytosis. Hence, it can be concluded that PECA nanoparticles comprise a promising formulation strategy for the peroral delivery of the chemical sterilitant D-Lys6-GnRH to the brushtail possum in New Zealand.

The hypothalamic-pituitary-ovarian axis and manipulations of the oestrous cycle in the brushtail possum.

The main purpose of this review is to provide a comprehensive update on what is known about the regulatory mechanisms of the hypothalamic-pituitary-ovarian axis in the brushtail possum, and to report on the outcomes of attempts made to manipulate by hormonal means, these processes in the possum. Over the last 15 years, several unique features of possum reproductive physiology have been discovered. These include an extended follicular phase despite elevated concentrations of FSH during the luteal phase, and early expression of LH receptors on granulosa cells of small antral follicles, suggesting a different mechanism for the selection of a dominant follicle. The use of routine synchronisation protocols that are effective in eutherians has failed to be effective in possums, and so the ability to reliably synchronise oestrus in this species remains a challenge.

Secretagogues stimulate electrogenic HCO3- secretion in the ileum of the brushtail possum, Trichosurus vulpecula: evidence for the role of a Na+/HCO3- cotransporter.

Fluid secretion is essential for intestinal function and, in eutherian mammals, is driven by electrogenic Cl(-) transport, which is dependent upon a bumetanide-sensitive, basolateral Na(+)/K(+)/2 Cl(-) cotransporter, NKCC1. However, ileal secretion in the brushtail possum, a marsupial, involves a fundamentally different process, since NKCC1 expression is low in this tissue and the secretagogue-induced short circuit current (I(sc)) is insensitive to bumetanide. In view of these differences we have investigated the basis of the secretory response of the possum ileum. In the Ussing chamber the secretory I(sc) is independent of Cl(-) but dependent upon Na(+) and serosal HCO(3)(-)/CO(2), suggesting that secretagogues stimulate electrogenic HCO(3)(-) secretion. In agreement with this, serosal DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonate; 1 mmol l(-1)) inhibited the secretory response. However, acetazolamide (1 mmol l(-1)) and serosal amiloride (1 mmol l(-1)) had little effect, indicating that HCO(3)(-) secretion is driven by HCO(3)(-) transport from the serosal solution into the cell, rather than hydration of CO(2) by carbonic anhydrase. Consistent with this the pancreatic variant of the electrogenic Na(+)/HCO(3)(-) cotransporter (pNBC) is highly expressed in the ileal epithelium and is located in the basolateral membrane of the epithelial cells, predominantly in the mid region of the villi, with lower levels of expression in the crypts and no expression in the villous tips. We conclude that the secretory response of the possum ileum involves electrogenic HCO(3)(-) secretion driven by a basolateral pNBC and that the ileal HCO(3)(-) secretion is associated with a specialised function of the possum ileum, most probably related to hindgut fermentation.

Physiology and pharmacology of the brushtail possum gastrointestinal tract: relationship to the human gastrointestinal tract.

Oral formulations are typically based on studies from eutherian animal models. This review introduces information relating to oral formulations for a marsupial species, the Australian brushtail possum (Trichosurus vulpecula) that has arisen from research into new methods for controlling this species - a major vertebrate pest in New Zealand. Morphologically, the gastrointestinal tract of the brushtail possum is similar to that of hindgut fermenting eutherian species, but there are some striking differences in function. Limited data suggests that the pharmacokinetics and bioavailability of administered drugs are similar to that in eutherian species, but there is some evidence that possums may have specific mechanisms for handling the intake of plant toxins and xenobiotics. The development of oral formulations for a free-ranging pest species presents several challenges above those encountered in the development of therapeutic formulations for humans and domestic animals. Use of a marsupial animal model may lead to new strategies for oral formulations in humans.

Seasonal changes in mesotocin and localization of its receptor in the prostate of the brushtail possum (Trichosurus vulpecula).

The prostate gland in the brushtail possum grows and regresses seasonally. It has similarities to the human prostate and may therefore provide a unique model for investigating prostatic hyperplasia. Oxytocin has been implicated in the regulation of prostate growth in eutherian mammals, and the initial aim of this study was to identify and localize the marsupial equivalent, mesotocin, and its receptor in the prostate of the brushtail possum. Seasonal changes in prostatic mesotocin concentrations and receptor localization were then assessed and related to prostate growth. Mesotocin and mesotocin receptor gene transcripts with high sequence homology to eutherian oxytocin/oxytocin receptors were demonstrated, and mesotocin, neurophysin, and the receptor were all localized predominantly in the epithelial cells of the glandular acini. Western blot analysis confirmed the presence of a single immunoreactive receptor protein of approximately 60 Mr-3. Prostatic mesotocin concentrations were highest immediately before the increases in prostate weight associated with the autumn and spring breeding periods. At this time, mesotocin receptors were also present in the prostatic capsule in addition to those present in the glandular tissue. Mesotocin concentrations proceeded to decrease in association with the regression of prostate size toward the end of the breeding periods. No significant differences were present in serum testosterone or dihydrotestosterone throughout the year. The identification of mesotocin and its receptor in the possum prostate and the demonstration of seasonal changes in local mesotocin concentrations preceding changes in prostate size suggests that mesotocin may play a physiological role in regulating prostate growth and regression.