Artemin-GFRα3 interactions partially contribute to acute inflammatory hypersensitivity.

The expression of artemin (ARTN), a glial cell line-derived neurotrophic factor (GDNF) family ligand, increases in pre-clinical models of nociception and recent evidence suggests this growth factor may play a causative role in inflammatory pain mechanisms. The aim of this study was to demonstrate functional inhibition of ARTN with monoclonal antibodies and to determine whether ARTN neutralisation could reverse inflammatory pain in mice. We show that monoclonal antibodies with high affinity to ARTN, completely inhibit ARTN-induced Ret and ERK activation in a human neuroblastoma cell line, and block capsaicin-induced CGRP secretion from primary rat DRG cultures. In addition, administration of anti-ARTN antibodies to mice provides a transient, partial reversal (41%) of FCA-induced mechanical hypersensitivity. Anti-ARTN antibodies had no effect on hypersensitivity in response to partial nerve ligation in mice. These data suggest that ARTN-GFRα3 interactions partially mediate early stage nociceptive signalling following an inflammatory insult.

Differential effects of repeated long and brief maternal separation on behaviour and neuroendocrine parameters in Wistar dams.

Repeated, prolonged maternal separation has been suggested to model the development of a depression-like syndrome in rats. The long separations from the pups have been proposed to be stressful for the dams, which in turn could mediate the changes seen in adult offspring. In the present study we investigated whether prolonged maternal separation really is stressful for rat dams by studying parameters known to be affected by long-term stress such as spontaneous motor activity, anxiety-like behaviour, adrenal gland weight and plasma corticosterone levels. Dams were separated from their litter for either 4 h (MS240) or 15 min (MS15) on eight random days during postnatal day 1-14, or left undisturbed (animal facility reared, AFR). After weaning MS240 dams showed decreased peripheral activity and habituated slower in horizontal activity. On the contrary, MS15 dams showed more peripheral activity and less rearing activity compared to both AFR and MS240 dams when habituated to the testing apparatus, suggesting that MS15 dams are more anxious. The adrenal glands from MS15 dams weighed significantly less and plasma corticosterone levels were significantly higher compared to AFR and MS240 dams. These results suggest that repeated brief maternal separations from pups could be stressful for rat mothers, whereas prolonged separations are not. Since these results are in contrast to the current notion that the short separation procedure may be considered as a safe milieu, whereas the prolonged separations have been suggested to be stressful for both dams and pups, further studies in this field are warranted.

Twice daily long maternal separations in Wistar rats decreases anxiety-like behaviour in females but does not affect males.

Prolonged daily separations of rat pups from their mother have been reported to increase anxiety-like behaviour in adult offspring. However, there are an increasing number of studies not showing this. It has been proposed that the effect of long maternal separation (LMS) is partly due to the disruption of maternal care caused by the separations. The aim of the present study was to investigate whether increasing the number of daily separations would produce more robust effects in the adult offspring on anxiety-like behaviour in the defensive withdrawal test, and on spontaneous motor activity. Since previous studies of LMS have revealed sex differences in behaviour, we included both males and females. In our separation paradigm we subjected rat pups to either two daily 3h maternal separations during the first 2 weeks postpartum (LMS), two daily 15 min maternal separations (brief maternal separations, BMS) during the same time period to control for the effects of handling, or to normal husbandry conditions. As adults we found no effects of this LMS paradigm in male rats, although BMS males showed a tendency toward decreased anxiety-like behaviour. In contrast, LMS females showed a decrease in anxiety-like behaviour. We also found significant sex differences that were most prominent in the LMS group, indicating that females are more sensitive to our maternal separation paradigm. The present study suggests that increasing the number of maternal separations does not increase anxiety-like behaviour in neither male nor female Wistar rats.

Oxytocin decreases corticosterone and nociception and increases motor activity in OVX rats.

OBJECTIVES: In the present study the effects of oxytocin administered subcutaneously (s.c.) or intravaginally (i.vag.) on spontaneous motor activity, nociceptive thresholds and plasma corticosterone levels were examined in female ovariectomized (OVX) rats. METHODS: Oxytocin (1 mg/kg s.c. or 100 microg i.vag.) was administered once a day for 10 days to OVX rats. Controls received saline s.c. or cellulose gel i.vag. Spontaneous motor activity was observed in an open-field arena, nociceptive thresholds were investigated by the tail-flick test, and corticosterone and oxytocin plasma levels were measured by radioimmunassay, 3, 4 and 5 days respectively, after the end of the treatment period. RESULTS: Both oxytocin administered s.c. and i.vag. increased forward locomotion (p<0.05) and nociceptive thresholds (p<0.05) significantly. In addition, oxytocin s.c. increased the amount of locomotor activity (p<0.05). Plasma corticosterone levels were decreased (p<0.05) and oxytocin levels were unchanged when measured 5 days after the last administration of oxytocin s.c. or i.vag. CONCLUSION: The present data indicate that oxytocin induces a spectrum of long-lasting effects in OVX rats, including an increase in spontaneous motor activity, elevation of nociceptive thresholds and decrease of corticosterone levels. Similar effects may be induced by estrogens. In addition, these data indicate that i.vag. administration of oxytocin may be used to induce oxytocin-mediated effects.

Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs.

A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.

Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex.

Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cellulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1Cel6A) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1Cel6A fusion protein had been previously applied. This showed that the SPA-CBM1Cel6A fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.

Structural basis for recognition by an in vitro evolved affibody.

The broad binding repertoire of antibodies has permitted their use in a wide range of applications. However, some uses of antibodies are precluded due to limitations in the efficiency of antibody generation. In vitro evolved binding proteins, selected from combinatorial libraries generated around various alternative structural scaffolds, are promising alternatives to antibodies. We have solved the crystal structure of a complex of an all alpha-helical in vitro selected binding protein (affibody) bound to protein Z, an IgG Fc-binding domain derived from staphylococcal protein A. The structure of the complex reveals an extended and complementary binding surface with similar properties to protein-antibody interactions. The surface region of protein Z recognized by the affibody is strikingly similar to the one used for IgG(1) Fc binding, suggesting that this surface contains potential hot-spots for binding. The implications of the selected affibody binding-mode for its application as a universal binding protein are discussed.

A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products.

An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag Z(SPA-1), enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag Z(SPA-1), was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (P(T7)) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His(6)) tag in frame with the Z(SPA-1) tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

Anti-idiotypic protein domains selected from protein A-based affibody libraries.

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.