Primordial germ cell expression of SSEA1 and DDX4 (VASA) in female Trichosurus vulpecula (Marsupialia) reveals conserved and unique molecular patterns during marsupial germ cell development.

Development of primordial germ cells (PGCs: precursors to adult gametes) is a key process in vertebrate sexual differentiation. Marsupials are ideal to investigate this phenomenon because much of PGC migration and development unusually occurs postnatally in pouch young. However, investigation of the molecular dynamics underpinning PGC development is restricted to one marsupial model species: the tammar wallaby (Macropus eugenii). Given the reproductive diversity among clades, marsupial PGCs likely exhibit diversity in molecular patterns that could help uncover their developmental dynamics. Here we characterise PGC marker expression (SSEA1 and DDX4) in developing ovaries of the brushtail possum, Trichosurus vulpecula. Female germ cells expressed DDX4 from 6 days postpartum (dpp) and almost all germ cells expressed DDX4 by meiosis (40 dpp), consistent with M. eugenii and eutherian mammals. In contrast, PGCs and oogonia expressed SSEA1 from 12 dpp, throughout proliferation and until entry into meiosis (40-63 dpp). SSEA1 expression was temporally distinct from that of M. eugenii, in which SSEA1 expression persists only until 14 dpp, indicating differential expression between marsupial species at equivalent stages of germ cell development. Hence, the molecular characteristics of M. eugenii germ cells cannot be assumed for all marsupials, as at least one key molecule exhibits species-specific expression.

In vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum (Trichosurus vulpecula).

Feline infectious peritonitis (FIP) is a systemic, fatal, viral-induced, immune-mediated disease of cats caused by feline infectious peritonitis virus (FIPV). Mefloquine, a human anti-malarial agent, has been shown to inhibit FIPV in vitro. As a first step to evaluate its efficacy and safety profile as a potential FIP treatment for cats, mefloquine underwent incubation in feline, canine and common brush-tailed possum microsomes and phase I metabolism cofactors to determine its rate of phase I depletion. Tramadol was used as a phase I positive control as it undergoes this reaction in both dogs and cats. Using the substrate depletion method, the in vitro intrinsic clearance (mean ± S.D.) of mefloquine by pooled feline and common brush-tailed possum microsomes was 4.5 ± 0.35 and 18.25 ± 3.18 µL/min/mg protein, respectively. However, phase I intrinsic clearance was too slow to determine with canine microsomes. Liquid chromatography-mass spectrometry (LC-MS) identified carboxymefloquine in samples generated by feline microsomes as well as negative controls, suggesting some mefloquine instability. Mefloquine also underwent incubation with feline, canine and common brush-tailed possum microsomes and phase II glucuronidative metabolism cofactors. O-desmethyltramadol (ODMT or M1) was used as a positive control as it undergoes a phase II glucuronidation reaction in these species. The rates of phase II mefloquine depletion by microsomes by all three species were too slow to estimate. Therefore mefloquine likely undergoes phase I hepatic metabolism catalysed by feline and common brush-tailed possum microsomes but not phase II glucuronidative metabolism in all three species and mefloquine is not likely to have delayed elimination in cats with clinically normal, hepatic function.

Intrinsic clearance rate of O-desmethyltramadol (M1) by glucuronide conjugation and phase I metabolism by feline, canine and common brush-tailed possum microsomes.

Quantitative aspects of in vitro phase II glucuronidative metabolism of O-desmethyltramadol (O-DSMT or M1), the active metabolite of the analgesic drug tramadol, by feline, canine and common brush-tailed possum hepatic microsomes are described.Whilst previous studies have focused on the phase I conversion of tramadol to M1, this is the first report in which the phase II glucuronidative metabolic pathway of M1 has been isolated by an in vitro comparative species study.Using the substrate depletion method, microsomal phase II glucuronidative in vitro intrinsic clearance (Clint) of M1 was determined.The in vitro Clint (mean ± SD) by pooled common brush-tailed possum microsomes was 9.9 ± 1.7 µL/min/mg microsomal protein whereas the in vitro Clint by pooled canine microsomes was 1.9 ± 0.07 µL/min/mg microsomal protein. The rate of M1 depletion by feline microsomes, as measured solely by high pressure liquid chromatography, was too slow to determine. Liquid chromatography-mass spectrometry identified O-DSMT glucuronide in samples generated from all three species' microsomes, although the amount detected under the feline condition was minimal.This study indicates that M1 likely undergoes in vitro phase II glucuronidation by canine and common brush-tailed possum microsomes and, to a minor extent, by feline microsomes. The rate of depletion of M1 by phase I metabolism was also undertaken.When incubated with phase I co-factors and common brush-tailed possum microsomes or canine microsomes, M1 had an in vitro Clint of 47.6 and 22.8 µL/min/mg microsomal protein, respectively. However, due to a lack of CYP2B-like activity in the feline liver, unsurprisingly, M1 did not deplete when incubated with feline microsomes. Consequently, major M1 elimination pathways, using feline microsomes, were not determined."

Uterine molecular changes for non-invasive embryonic attachment in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae).

Pregnancy in mammals requires remodeling of the uterus to become receptive to the implanting embryo. Remarkably similar morphological changes to the uterine epithelium occur in both eutherian and marsupial mammals, irrespective of placental type. Nevertheless, molecular differences in uterine remodeling indicate that the marsupial uterus employs maternal defences, including molecular reinforcement of the uterine epithelium, to regulate embryonic invasion. Non-invasive (epitheliochorial) embryonic attachment in marsupials likely evolved secondarily from invasive attachment, so uterine defences in these species may prevent embryonic invasion. We tested this hypothesis by identifying localization patterns of Talin, a key basal anchoring molecule, in the uterine epithelium during pregnancy in the tammar wallaby (Macropus eugenii; Macropodidae) and the brush tail possum (Trichosurus vulpecula; Phalangeridae). Embryonic attachment is non-invasive in both species, yet Talin undergoes a clear distributional change during pregnancy in M. eugenii, including recruitment to the base of the uterine epithelium just before attachment, that closely resembles that of invasive implantation in the marsupial species Sminthopsis crassicaudata. Basal localization occurs throughout pregnancy in T. vulpecula, although, as for M. eugenii, this pattern is most specific prior to attachment. Such molecular reinforcement of the uterine epithelium for non-invasive embryonic attachment in marsupials supports the hypothesis that less-invasive and non-invasive embryonic attachment in marsupials may have evolved via accrual of maternal defences. Recruitment of basal molecules, including Talin, to the uterine epithelium may have played a key role in this transition.

cAMP-dependent secretagogues stimulate the NaHCO3 cotransporter in the villous epithelium of the brushtail possum, Trichosurus vulpecula.

In the ileum of the brushtail possum, Trichosurus vulpecula, fluid secretion appears to be driven by electrogenic HCO3- secretion. Consistent with this, the cystic fibrosis transmembrane conductance regulator is expressed in the apical membrane of the ileal epithelial cells and the pancreatic or secretory variant of the NaHCO3 cotransporter in the basolateral membrane. This suggests that in the possum ileum, electrogenic HCO3- secretion is driven by basolateral NaHCO3 cotransporter (NBC) activity. To determine if the NBC contributes to HCO3- secretion in the possum ileum, intracellular pH (pHi) measurements in isolated villi were used to demonstrate NBC activity in the ileal epithelial cells and investigate the effect of cAMP-dependent secretagogues. In CO2/HCO3--free solutions, recovery of the epithelial cells from an acid load was Na+-dependent and ≈80% inhibited by ethyl-isopropyl-amiloride (EIPA, 10 µmol L-1), indicative of the presence of an Na+/H+ exchanger, most likely NHE1. However, in the presence of CO2/HCO3-, EIPA only inhibited ≈ 50% of the recovery, the remainder was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 500 µmol L-1), indicative of NBC activity. Under steady-state conditions, NHE1 inhibition by EIPA had little effect on pHi in the presence or absence of secretagogues, but NBC inhibition with DIDS resulted in a rapid acidification of the cells, which was increased fivefold by secretagogues. These data demonstrate the functional activity of an NaHCO3 cotransporter in the ileal epithelial cells. Furthermore, the stimulation of NBC activity by secretagogues is consistent with the involvement of an NaHCO3 cotransporter in electrogenic HCO3- secretion.

The Use of Polyethylene Glycol in Mammalian Herbivore Diet Studies: What Are We Measuring?

Polyethylene glycol (PEG) has been used to study the intake and digestion of tannin-rich plants by mammalian herbivores because it preferentially binds to tannins. However, it is not clear whether the responses of herbivores to dietary PEG is due to increased protein availability from the release of tannin-bound protein, amelioration of tannin effects, or whether PEG also may bind to other compounds and change their activity in the gut. We used three native New Zealand tree species to measure the effect of PEG on the amount of foliage eaten by invasive common brushtail possums (Trichosurus vulpecula) and on in vitro digestible nitrogen (available N). The addition of PEG increased the in vitro available N content of Weinmannia racemosa foliage, and possums ate significantly more PEG-treated foliage than untreated foliage. However, possums also ate more PEG-treated Fuchsia excorticata foliage, even though PEG did not increase in vitro available N in this species. Possums ate very little Melicytus ramiflorus, regardless of PEG treatment, even though M. ramiflorus contained the highest concentration of in vitro available N. These results prompted us to use PEG and a protein supplement, casein, to manipulate the available N concentration of diets containing ground eucalypt foliage, a well-studied food species for possums. Again, the response of possums to PEG was independent of changes in in vitro available N. In addition, altering the protein content of the diet via the addition of casein did not affect how much food the possums consumed. We conclude that the effects of PEG on dry matter intake by mammalian herbivores are not due solely to the release of tannin-bound protein. There is need for a better understanding of PEG-tannin interactions in order to ensure that the use of PEG in nutritional studies does not outstrip an understanding of its mechanisms of action.

Variations in epithelial Na(+) transport and epithelial sodium channel localisation in the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, during the oestrous cycle.

The fluid in the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, is copious at ovulation when it may be involved in sperm transport or maturation, but is rapidly reabsorbed following ovulation. We have used the Ussing short-circuit current (Isc) technique and measurements of transcript and protein expression of the epithelial Na(+) channel (ENaC) to determine if variations in electrogenic Na(+) transport are associated with this fluid absorption. Spontaneous Isc (<20µAcm(-2) during anoestrus, 60-80µAcm(-2) in cycling animals) was inhibited by serosal ouabain. Mucosal amiloride (10µmolL(-1)), an inhibitor of ENaC, had little effect on follicular Isc but reduced luteal Isc by ~35%. This amiloride-sensitive Isc was dependent on mucosal Na(+) and the half-maximal inhibitory concentration (IC50)-amiloride (0.95µmolL(-1)) was consistent with ENaC-mediated Na(+) absorption. Results from polymerase chain reaction with reverse transcription (RT-PCR) indicate that αENaC mRNA is expressed in anoestrous, follicular and luteal phases. However, in follicular animals αENaC immunoreactivity in epithelial cells was distributed throughout the cytoplasm, whereas immunoreactivity was restricted to the apical pole of cells from luteal animals. These data suggest that increased Na(+) absorption contributes to fluid absorption during the luteal phase and is regulated by insertion of ENaC into the apical membrane of cul-de-sac epithelial cells.

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.

Photoreceptor topography and spectral sensitivity in the common brushtail possum (Trichosurus vulpecula).

Marsupials are believed to be the only non-primate mammals with both trichromatic and dichromatic color vision. The diversity of color vision systems present in marsupials remains mostly unexplored. Marsupials occupy a diverse range of habitats, which may have led to considerable variation in the presence, density, distribution, and spectral sensitivity of retinal photoreceptors. In this study we analyzed the distribution of photoreceptors in the common brushtail possum (Trichosurus vulpecula). Immunohistochemistry in wholemounts revealed three cone subpopulations recognized within two spectrally distinct cone classes. Long-wavelength sensitive (LWS) single cones were the largest cone subgroup (67-86%), and formed a weak horizontal visual streak (peak density 2,106 ± 435/mm2) across the central retina. LWS double cones were strongly concentrated ventrally (569 ± 66/mm2), and created a "negative" visual streak (134 ± 45/mm2) in the central retina. The strong regionalization between LWS cone topographies suggests differing visual functions. Short-wavelength sensitive (SWS) cones were present in much lower densities (3-10%), mostly located ventrally (179 ± 101/mm2). A minority population of cones (0-2.4%) remained unlabeled by both SWS- and LWS-specific antibodies, and may represent another cone population. Microspectrophotometry of LWS cone and rod visual pigments shows peak spectral sensitivities at 544 nm and 500 nm, respectively. Cone to ganglion cell convergences remain low and constant across the retina, thereby maintaining good visual acuity, but poor contrast sensitivity during photopic vision. Given that brushtail possums are so strongly nocturnal, we hypothesize that their acuity is set by the scotopic visual system, and have minimized the number of cones necessary to serve the ganglion cells for photopic vision.

In vitro hepatic microsomal metabolism of meloxicam in koalas (Phascolarctos cinereus), brushtail possums (Trichosurus vulpecula), ringtail possums (Pseudocheirus peregrinus), rats (Rattus norvegicus) and dogs (Canis lupus familiaris).

Quantitative and qualitative aspects of in vitro metabolism of the non-steroidal anti-inflammatory drug meloxicam, mediated via hepatic microsomes of specialized foliage (Eucalyptus) eating marsupials (koalas and ringtail possums), a generalized foliage eating marsupial (brushtail possum), rats, and dogs, are described. Using a substrate depletion method, intrinsic hepatic clearance (in vitro Clint) was determined. Significantly, rates of oxidative transformation of meloxicam, likely mediated via cytochromes P450 (CYP), were higher in marsupials compared to rats or dogs. The rank order of apparent in vitro Clint was brushtail possums (n=3) (mean: 394µL/min/mg protein), >koalas (n=6) (50), >ringtail possums (n=2) (36) (with no significant difference between koalas and ringtail possums), >pooled rats (3.2)>pooled dogs (in which the rate of depletion, as calculated by the ratio of the substrate remaining was <20% and too slow to determine). During the depletion of meloxicam, at a first-order rate constant, 5-hydroxymethyl metabolite (M1) was identified in the brushtail possums and the rat as the major metabolite. However, multiple hydroxyl metabolites were observed in the koala (M1, M2, and M3) and the ringtail possum (M1 and M3) indicating that these specialized foliage-eating marsupials have diverse oxidation capacity to metabolize meloxicam. Using a well-stirred model, the apparent in vitro Clint of meloxicam for koalas and the rat was further scaled to compare with published in vivo Cl. The closest in vivo Cl prediction from in vitro data of koalas was demonstrated with scaled hepatic Cl(total) (average fold error=1.9) excluding unbound fractions in the blood and microsome values; whereas for rats, the in-vitro scaled hepatic Cl fu(blood, mic), corrected with unbound fractions in the blood and microsome values, provided the best prediction (fold error=1.86). This study indicates that eutherians such as rats or dogs serve as inadequate models for dosage extrapolation of this drug to marsupials due to differences in hepatic turnover rate. Furthermore, as in vivo Cl is one of the pharmacokinetic indexes for determining therapeutic drug dosages, this study demonstrates the utility of in vitro to in vivo scaling as an alternative prediction method of drug Cl in koalas.

A faecal index of diet quality that predicts reproductive success in a marsupial folivore.

Estimating the nutritional value of a herbivore's diet is difficult because it requires knowing what the animal eats, the relative quality of each component and how these components interact in relation to animal physiology. Current methods are cumbersome and rely on many assumptions that are hard to evaluate. We describe a new method for estimating relative diet quality directly from faeces that avoids the problems inherent in other methods. We combine this method with near infrared reflectance spectroscopy (NIRS) to analyse many samples and thus provide a technique with immense value in ecological studies. The method stems from the correlation between the concentrations of dietary and faecal nitrogen in herbivores eating a tannin-free diet, but a weaker relationship in browsers that ingest substantial amounts of tannins, which form complexes with proteins. These complexes reduce the availability of nitrogen and may increase faecal nitrogen concentrations. Using the tannin-binding compound, polyethylene glycol, we showed that tannin-bound nitrogen is a significant and variable part of faecal nitrogen in wild common brushtail possums (Trichosurus vulpecula). We developed a technique to measure faecal available nitrogen and found that it predicted the reproductive success of female brushtail possums in northern Australia. Faecal available nitrogen combined with NIRS provides a powerful tool for estimating the relative nutritional value of the diets of browsing herbivores in many ecological systems. It is a better indicator of diet quality than other commonly used single-nutrient measures such as faecal nitrogen and foliage analysis paired with observed feeding behaviour.

Scent chemicals of the brushtail possum, Trichosurus vulpecula.

The common brushtail possum (Trichosurus vulpecula) is the most widespread browsing marsupial in Australia, where it occupies woodland, agricultural, and urban environments. Following its introduction into New Zealand in the 19th century it has become a major feral pest, threatening native forests. The adaptability of the possum is thought to be due in part to its social organization, in which chemical communication is important. Possums have cloacal glands and exhibit related marking behavior. This study sought to characterize the chemicals involved in scent marking. Swabs were taken of the cloacal region of 15 possums (5 females, 10 males) from north-eastern Tasmania and analyzed by gas chromatography-mass spectrometry. There was a large number of compounds present, including 81 branched and unbranched, and saturated and unsaturated, fatty acids (C(4)-C(15)) and alcohols (C(6)-C(26)); 27 esters of 2,6- and 2,7-dimethyloctanol; 29 esters of formic acid; 39 sulfur compounds including S(8) and a series of dialkyl disulfides, trisulfides, and tetrasulfides (C(4)-C(10)); and several alkylglycerol ethers. Many of these cloacal compounds are new to biology. There was considerable individual variability in the relative amounts of compounds found, and no evident sex differences, although the study was not designed to test this. This pattern suggests that these compounds may be acting collectively as a signature mixture of semiochemicals, carrying information on the individual, its kinship, and physiological and social status. This is the first detailed description of putative semiochemicals in any marsupial species.

Bait aggregation to reduce cost and toxin use in aerial 1080 baiting of small mammal pests in New Zealand.

BACKGROUND: The metabolic toxin sodium fluoroacetate ('compound 1080') is widely used for controlling introduced mammalian pests in New Zealand. For large-scale operations, 1080 is distributed aerially in bait to kill brushtail possums (Trichosurus vulpecula Kerr) and ship rats (Rattus rattus L.). While usually successful in reducing pest populations by > 80%, widespread distribution of toxic bait is relatively expensive and raises concerns from some members of the public. Here, trials with spatial aggregation of baits in forested habitats were conducted to determine whether this can reduce toxin usage while maintaining operational efficacy. RESULTS: When 1080 baits were aggregated into clusters (by hand sowing) or into strips (by precision aerial deployment), indices of possum relative abundance were reduced by 92-100%, compared with 73-100% reductions using conventional aerial broadcasting, while all methods reduced relative abundance indices of rats by 88% or greater. Radio tracking indicated a kill rate of > 90% against possums, regardless of bait distribution method. CONCLUSIONS: Simply by modifying bait distribution patterns, spatial aggregation can be used to maintain the high encounter rate of pests with 1080 bait that is necessary for operational efficacy, while reducing current toxin usage by up to 80%. Aggregated bait delivery could have relevance for other mammalian pest control scenarios internationally.

CFTR is restricted to a small population of high expresser cells that provide a forskolin-sensitive transepithelial Cl- conductance in the proximal colon of the possum, Trichosurus vulpecula.

The cystic fibrosis transmembrane conductance regulator (CFTR) is central to anion secretion in both the possum and eutherian small intestine. Here, we investigated its role in the possum proximal colon, which has novel transport properties compared with the eutherian proximal colon. Despite considerable CFTR expression, high doses of the CFTR activator forskolin (EC(50)≈10 µmol l(-1)) were required for a modest, CFTR-dependent increase in short-circuit current (I(sc)) in the proximal colon. Presumably, this is because CFTR is restricted to the apical membrane of a small population of CFTR high expresser (CHE) cells in the surface and upper crypt epithelium. Furthermore, although the forskolin-stimulated I(sc) was dependent on serosal Na(+), Cl(-) and HCO(3)(-), consistent with anion secretion, inhibition of the basolateral Na-K-2Cl(-) (NKCC1) or Na-HCO(3) (pNBCe1) cotransporters did not prevent it. Therefore, although NKCC1 and pNBCe1 are expressed in the colonic epithelium they do not appear to be expressed in CHE cells. At low doses (IC(50)≈1 µmol l(-1)), forskolin also decreased the transepithelial conductance (G(T)) of the colon through inhibition of a 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid-sensitive anion conductance in the basolateral membrane of the CHE cells. This conductance is arranged in series with CFTR in the CHE cells and, therefore, the CHE cells provide a transepithelial Cl(-) conductance for passive Cl(-) absorption across the epithelium. Inhibition of the basolateral Cl(-) conductance of the CHE cells by forskolin will inhibit Na(+) absorption by restricting the movement of its counter-ion Cl(-), assisting in the conversion of the tissue from an absorptive to a secretory state.

Seasonal changes in morphology and steroid receptor expression in the prostate of the brushtail possum (Trichosurus vulpecula): an animal model for the study of prostate growth?

The prostate of the brushtail possum undergoes growth and regression during the year. The present study investigated the morphological changes and expression of androgen and oestrogen receptors during the breeding and non-breeding seasons. Prostate tissue was collected from adult possums at 2-monthly intervals. The periurethral and outer glandular areas were separated and the volume of stromal, epithelial and luminal tissues measured in each area. Immunohistochemistry was used to investigate cell proliferation with proliferating cell nuclear antigen (PCNA) and to localise androgen receptor (AR) and oestrogen receptors α and ß (ERα, ERß). Seasonal changes in expression of the three receptors were investigated using quantitative PCR and western blot analysis. During the breeding season the volume of stromal tissue in the periurethral area and the luminal volume in the glandular area significantly increased. The change in periurethral volume was associated with increased PCNA-immunopositive cells. While the localisation of AR to the stromal and epithelial cells did not change, there was a significant increase in receptor expression before the main breeding season. ERα and ERß expression and localisation did not alter during the year. Similarities in receptor expression and localisation suggest that the possum may be a suitable animal model for the study of human prostate growth.

The distribution and expression of CFTR restricts electrogenic anion secretion to the ileum of the brushtail possum, Trichosurus vulpecula.

In eutherian mammals, fluid secretion is essential for intestinal function. This is driven by electrogenic Cl(-) secretion, which involves a NaK2Cl cotransporter (NKCC1) in the enterocyte basolateral membrane and the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, in the possum ileum, NKCC1 expression is low and secretagogues stimulate electrogenic HCO(3)(-) secretion driven by a basolateral NaHCO(3) cotransporter (pNBCe1). Here we investigated whether electrogenic anion secretion occurs in possum duodenum and jejunum and determined the role of CFTR in possum intestinal anion secretion. Prostaglandin E(2) (PGE(2)) and forskolin stimulated a large increase in ileal short-circuit current (I(sc)), consistent with electrogenic HCO(3)(-) secretion, but had little effect on the duodenal and jejunal I(sc). Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH101) inhibited cloned possum CFTR in cultured cells and the PGE(2)-stimulated ileal I(sc), implicating CFTR in ileal HCO(3)(-) secretion. Consistent with this, CFTR is expressed in the apical membrane of ileal crypt and lower villous cells, which also express pNBCe1 in the basolateral membrane. In contrast, duodenal and jejunal CFTR expression is low relative to the ileum. Jejunal pNBCe1 expression is also low, whereas duodenal and ileal pNBCe1 expression are comparable. All regions have low NKCC1 expression. These results indicate that cAMP-dependent electrogenic Cl(-) secretion does not occur in the possum small intestine because of the absence of CFTR and NKCC1. Furthermore, CFTR functions as the apical anion conductance associated with HCO(3)(-) secretion and its distribution limits electrogenic HCO(3)(-) secretion to the ileum.

Energy substrate utilization in the common brushtailed possum (Trichosurus vulpecula) using intravenous tolerance tests.

The aim of this study was to investigate the energy substrate requirements of the common brushtailed possum (Trichosurus vulpecula) using intravenous tolerance tests for glucose, alanine, and propionate in five adult male and female animals under standardized conditions. Significant differences (p<0.01) were observed for fasting blood glucose values between males (6.3±0.16 mmol L(-1)) and females (4.8±0.13 mmol L(-1)), and males had a significantly (p<0.001) increased response to glucose. All animals returned to fasting glucose levels within 120 min after the glucose challenge. No significant change in blood glucose levels was observed for either the alanine or propionate tolerance tests (p>0.05). However, following propionate administration, there was a highly significant (p<0.001) decrease in blood lactate concentrations over 120 min. There was no evidence of ketone formation using ß-hydroxybutyrate as a biomarker during any of the tests, indicating that there was no significant switch to lipolysis. In conclusion, the study provides new information on energy substrate utilization in this species and has identified that a gluconeogenic response normally identified in other species is not apparent in the common brushtailed possum.

Prolactin acts on the hypothalamic-pituitary axis to modulate follicle-stimulating hormone gene expression in the female brushtail possum (Trichosurus vulpecula).

Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic-pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 µg) resulted in an 18-fold stimulation (P<0.05) of progesterone (P(4)) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 (n=10) or saline (n=8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable (P<0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation (P<0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P(4) concentrations were not different (P=0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.

Vertebrate pesticide risk assessment by indigenous communities in New Zealand.

In New Zealand, the vertebrate pesticide sodium fluoroacetate (Compound 1080) is aerially applied in baits for control of the brush-tailed possum Trichosurus vulpecula (Kerr, 1792). Maori, the indigenous people of New Zealand, have raised concerns about 1080 impacts on culturally-important species. Here, we outline two steps taken to help Maori assess 1080 risk. First, field research was undertaken to determine if naturally-occurring plants utilized by a Maori community for food and medicine would take up 1080 from baits. Single baits were placed at the base of individual plants of two species, pikopiko (Asplenium bulbiferum) and karamuramu (Coprosma robusta). Plants were sampled at various times up to 56 days, and samples were analyzed for 1080 content. No 1080 was detected in any of the pikopiko samples, whereas 1080 was detected in karamuramu, at a maximum concentration of 5 ppb after seven days, and 2.5 ppb after 14 days. This concentration decreased to 0 at 28 days, indicating that 1080 was not persistent. The results of the present study suggest there is negligible risk of humans being poisoned by consuming plants that have taken up 1080 from baits. To allay community concerns that minute concentrations of 1080 might influence the medicinal properties of plants, it is suggested that a withholding period of 30 days after 1080 control operations could be adopted. Second, after further consultation we undertook a review of the scientific literature relating to 1080 impacts on additional non-target species of cultural importance to Maori. The information was presented on an interactive foodweb database that allowed the collection and presentation of a large volume of complex information about 1080 in a holistic and pictorial fashion. This database was presented to many Maori communities throughout New Zealand, and feedback was overwhelmingly positive. The database is likely to play a key role in informing these communities about 1080, and is seen as an important new tool to help these communities make their own risk assessments.