High-Affinity Receptor Sites and Rapid Proteolytic Inactivation of Neurotensin in Primary Cultured Neurons

The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37 degrees C of [monoiodo-Tyr3]neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preparations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7-Arg8, Arg8-Arg9, and Pro10-Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7-Arg8 bond and contributes to the hydrolysis mainly at the Pro10-Tyr11 site. However, the latter breakdown is also generated by a neurotensin-degrading neutral metallopeptidase. The cleavage at the Arg8-Arg9 bond is due to a peptidase that can be specifically inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanyl-p- aminobenzoate. The secondary processing occurring on neurotensin degradation products are: a bestatin-sensitive aminopeptidasic conversion of neurotensin11-13 to free Tyr11, and a rapid cleavage of neurotensin8-13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.     

Ultrastructure of oogenesis in the brushtail possum

Marsupial ova are slightly larger on average than those of eutherians. They have often been described as "yolky", although the nature and roles of their contents in later development are poorly understood. In the ova of all marsupial species studied, large quantities of electron-lucent vesicles are present. This study describes the ultrastructure of oogenesis in the brushtail possum, Trichosurus vulpecula. Oocytes of primordial follicles contain a variety of organelles of unknown function, some of which have been described previously in bandicoots. Large electron-lucent vesicles begin to accumulate after the transition from primordial follicle to primary follicle and are derived at least partly from the Golgi and endoplasmic reticulum. Other inclusions that are absent in some other marsupial species are present in growing possum oocytes in smaller quantities, including yolklike lipid bodies and electron-dense granular bodies.     

Xenobiotic metabolism in Australian marsupials

The Australian marsupials are significant and unique Australian fauna. Xenobiotic metabolism is the process of enzymatic modification of xenobiotics, which include the chemicals, such as agricultural chemicals and natural dietary toxins, that these animals may be exposed to. Very little is known about the enzymes involved in xenobiotic metabolism in this unique group of animals. Folivore marsupials such as the koala (Phascolarctos cinereus and the brushtail possum (Trichosurus vulpecula) represent unique adaptation which has only been relatively superficially examined to date. We provide an overview of our current knowledge of marsupial xenobiotic metabolism.     

cDNA cloning of luteinizing hormone subunits from brushtail possum and red kangaroo

Luteinizing hormone (LH) plays an important role in the reproductive cycles of all mammals. There is a large amount of both nucleotide and amino acid sequence data available for LH from eutherian mammals, but little is known about the primary structure of LH in marsupials. We have used consensus PCR primers to generate specific probes for screening pituitary cDNA libraries and report the cloning of the cDNAs encoding the α-subunit of LH (also shared by a number of other glycoprotein hormones) and the LH-specific β-subunit, from the common brushtail possum, Trichosurus vulpecula, and the red kangaroo, Macropus rufus. Southern blotting experiments indicated that both genes are probably present as single copies. Comparison of the deduced marsupial protein sequences with homologous sequences from other vertebrates revealed a high degree of conservation, especially for the α-subunit. These sequences represent the first complete primary structures for a marsupial glycoprotein hormone to have been elucidated. Received: 30 January 1998 / Accepted: 10 April 1998     

In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers. This epididymal cell and sperm co-culture system for marsupial species may facilitate the identification of specific epithelial factors that affect sperm maturation, particularly in a species in which morphological maturation is readily visible.     

High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

Novel alleles in classical major histocompatibility complex class II loci of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

We have investigated the diversity of class II major histocompatibility complex (MHC) loci in the brushtail possum (Trichosurus vulpecula), an important marsupial pest species in New Zealand. Immunocontraceptive vaccines, a method of fertility control that employs the immune system to attack reproductive cells or proteins, are currently being researched as a means of population control for the possum. Variation has been observed in the immune response of individual possums to immunocontraceptives. If this variability is under genetic control, it could compromise vaccine efficacy through preferential selection of animals that fail to mount a significant immune response and remain fertile. The MHC is an important immune region for antigen presentation and as such may influence the response to immunocontraceptives. We used known marsupial MHC sequences to design polymerase chain reaction primers to screen for possum MHC loci. Alpha and beta chains from two class II families, DA and DB, were found in possums throughout New Zealand. Forty new class II MHC alleles were identified in the possum, and the levels of variability in the MHC of this marsupial appear to be comparable to those of eutherian species. Preliminary population surveys showed evidence of clustering/variability in the distribution of MHC alleles in geographically separate locations. The extensive variation demonstrated in possums reinforces the need for further research to assess the risk that such MHC variation poses for long-term immunocontraceptive vaccine efficacy.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Identification of novel major histocompatibility complex class I sequences in a marsupial,the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The major histocompatibility complex (MHC) is an essential part of the vertebrate immune response. MHC genes may be classified as classical, non-classical or non-functional pseudogenes. We have investigated the diversity of class I MHC genes in the brushtail possum, a marsupial native to Australia and an introduced pest in New Zealand. The MHC of marsupials is poorly characterised compared to eutherian mammal species. Comparisons between marsupials and eutherians may enhance understanding of the evolution and functions of this important genetic region. We found a high level of diversity in possum class I MHC genes. Twenty novel sequences were identified using polymerase chain reaction (PCR) primers designed from existing marsupial class I MHC genes. Eleven of these sequences shared a high level of homology with the only previously identified possum MHC class I gene TrvuUB and appear to be alleles at a single locus. Another seven sequences are also similar to TrvuUB but have frame-shift mutations or stop codons early in their sequence, suggesting they are non-functional alleles of a pseudogene locus. The remaining sequences are highly divergent from other possum sequences and clusters with American marsupials in phylogenetic analysis, indicating they may have changed little since the separation of Australian and American marsupials.     

Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum)

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N‐linked glycosylation sites, a potential N‐terminal signal peptide and a potential C‐terminal trans‐membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N‐terminal signal peptide sequences and C‐terminal trans‐membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae. Mol. Reprod. Dev. 52:174–182, 1999. © 1999 Wiley‐Liss, Inc.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

Isolation,structure and biologic activity of chicken intestinal neurotensin

Using a radioimmunoassay towards bovine neurotensin (NT), chicken NT has been purified to homogeneity from extracts of intestine and its amino acid sequence determined to be: <Glu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu-OH. The molecule is identical to the bovine peptide except for the 3 amino acid substitutions located in its NH₂-terminal half and italicized above (His/Tyr; Val/Glu; Ala/Pro). The structure for chicken NT is consistent with earlier immunochemical studies which indicated a COOH-terminal homology with bovine NT [1]. The peptide isolated was shown to be near equipotent with bovine NT in its ability to induce hypotension, hyperglycemia, and cyanosis in the anesthesized rat, underscoring the importance of the COOH-terminal residues in NT for biological activity.     

Microsatellite markers for the Common ringtail possum (Pseudocheirus peregrinus) and their amplification in other Pseudocheirids

Eleven microsatellite markers were developed for the Common ringtail possum, an arboreal marsupial abundant in fragmented forests of south-eastern Australia. Loci were highly polymorphic (4-32 alleles per locus) and heterozygosity ranged from 0.66 to 1. Two loci deviated significantly from Hardy-Weinberg equilibrium proportions, possibly because of low-frequency null alleles. These markers will be informative for examining patterns of gene flow, relatedness and mating systems within fragmented populations of the Common ringtail possum and have potential for use in other Pseudocheirids.     

Isolation and characterization of microsatellite loci from the Australian endemic mountain pygmy‐possum,Burramys parvus Broom

The endemic mountain pygmy‐possum, Burramys parvus, is an endangered Australian marsupial restricted to mainland alpine regions. Population structure, breeding system and gene flow are crucial for the development of effective conservation strategies, but have not been investigated in B. parvus. Here we have isolated eight polymorphic microsatellite markers from B. parvus to investigate these parameters. We found two to 12 alleles with observed heterozygosity values ranging from 0.321 to 0.878 for these loci in initial estimates from a single population.     

Cloning and characterisation of a zona pellucida 3 cDNA from a marsupial, the brushtail possum Trichosurus vulpecula

The mammalian zona pellucida (ZP) is an extracellular glycoprotein coat that plays vital roles throughout fertilisation and preimplantation development. Like that of eutherian mammals the brushtail possum ZP is composed of three glycosylated proteins of 137 kDa, 92 kDa and 62 kDa. The 62 kDa protein is a ZP3 orthologue based on its nucleotide and deduced amino acid sequence. The brushtail possum ZP3 cDNA isolated in this study is 1305 nucleotides with an open reading frame encoding a 422 amino acid peptide of 45.7 kDa. Possum ZP3 has a 46% amino acid identity with eutherian ZP3 and shares similar structural characteristics including 12 conserved cysteine residues, N-linked glycosylation sites and hydrophobic regions. Like human and rabbit ZP1 an altered furin cleavage site upstream of the C-terminal hydrophobic domain also occurs in possum ZP3 (S-R-K-R), suggestive of processing by a furin-related endoprotease. Expression of brushtail possum ZP3 is limited to the ovary. Characterisation of brushtail possum ZP3 will enable examination of its functional role in marsupial fertilisation and its effectiveness as an immunocontraceptive agent.     

Degradation of neurotensin by rabbit brain endo-oligopeptidase A and endo-oligopeptidase B (proline-endopeptidase)

The degradation of neurotensin and D-Tyr11 neurotensin by apparently homogeneous preparations of rabbit brain endo-oligopeptidase A and endo-oligopeptidase B (Proline-endopeptidase) was studied. Peptide fragments were isolated by high performance liquid chromatography and identified by amino acid analysis. Endo-oligopeptidase A cleaved neurotensin at the Arg8-Arg9 bond whereas D-Tyr11 neurotensin was not significantly hydrolysed. Endo-oligopeptidase B cleaved at the carboxyl side of Pro7, Pro10 in neurotensin and at Pro7 in D-Tyr11 neurotensin. The concentration dependent inhibition of neurotensin degradation by bradykinin and vice-versa represents additional evidence that endo-oligopeptidase A cleaves both Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin.     

Two-dimensional proton NMR studies on poly(VPGVG) and its cyclic conformational correlate, cyclo(VPGVG)3

Two-dimensional nuclear Overhauser enhancement (2D NOESY) data are reported for the polypentapeptide of elastin, poly(VPGVG), and the cyclopentadecapeptide, cyclo(VPGVG)3. In both, the repeating type II Pro2-Gly3 beta-turn can be derived from the NOE data, providing confirmation of many previous studies. In addition, other through-space connectivities are detailed that also compare favorably with previously determined crystal and solution structures for cyclo(VPGVG)3. Also, near identical data for the cyclopentadecapeptide and the polypentapeptide demonstrate the cyclic conformation-linear (helical) conformational correlate relationship between the two molecules. The 2D NOESY experiment is seen to be an effective means of establishing the presence or absence of a conformational relationship between a cyclic repeating sequence and its higher molecular weight linear counterpart. This is an approach of substantial practical value when developing the conformation of sequential polypeptides and when attempting to identify the presence of the conformation of a repeating peptide sequence within a more complex primary structure. Having established the basic conformational relationship between a cyclic conformation and its linear helical counterpart, cross peaks present in the linear helical structure that are not present in the cyclic conformational correlate can provide information on the interactions between adjacent turns of the helix. In this connection, a Val gamma CH3 in equilibrium Pro beta CH2 interaction is reported that can be the basis for determining the number of pentamers per turn of helix once it is determined whether it is dominantly the Val1 or Val4 gamma CH3 that is interacting with the Pro2 beta CH2.