Eugenin An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii Russell V. Baudinette 1, *, Pinmanee Boontheung 2 , Ian F. Musgrave 3 , Paul A. Wabnitz 2 , Vita M. Maselli 2 , Jayne Skinner 1 , Paul F. Alewood 4 , Craig S. Brinkworth 2 and John H. Bowie 2 1 Department of Environmental Biology, The University of Adelaide, South Australia 2 Department of Chemistry, The University of Adelaide, South Australia 3 Department of Clinical and Experimental Pharmacology, The University of Adelaide, South Australia 4 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia Marsupials are born in an immature state and many of the developmental processes that occur in these mammals take place during pouch life [1]. After a short period of intrauterine development, the young marsupial crawls unaided to the mother’s pouch, atta- ches to a teat, and undergoes further development in an aerial environment [2] (Fig. 1). The pouch microcli- mate is characterized by high humidity, and a constant temperature close to maternal body temperatures [3]. The pouch, with its warm, moist environment, is a favourable environment for microorganisms. It has been shown that a variety of Gram-positive bacilli are present in marsupial pouches, together with lesser amounts of Gram-negative bacilli [4,5]. The bacterial content of the pouch diminishes significantly upon arrival and occupancy of the young marsupial [6]. When the young first crawls into the pouch, it has essentially no immune system of its own and must rely on that provided by the mother [1,7], even though it has been reported that an immunoglobulin is present in fetal and newborn sera of the Tammar wallaby (Macropus eugenii) [7]. With increasing devel- opment, the young produces its own immune system. For example, T and B cells are first detected 50 days into the development of the young wallaby in the pouch [7], and it has been shown that cholecystokinin 8 (CCK8) (a neuropeptide which engenders T and B cell proliferation) is present in the brains of mature Keywords eugenin; immunomodulator; lactating female; Tammar wallaby (Macropus eugenii) Correspondence J. H. Bowie, Department of Chemistry, The University of Adelaide, South Australia, 5005 Fax: +61 08 83034358 Tel: +61 08 83035767 E-mail: john.bowie@adelaide.edu.au *Author deceased (Received 30 August 2004, revised 18 October 2004, accepted 16 November 2004) doi:10.1111/j.1742-4658.2004.04483.x Eugenin [pGluGlnAspTyr(SO 3 )ValPheMetHisProPhe-NH 2 ] has been isolated from the pouches of female Tammar wallabies (Macropus eugenii) carrying young in the early lactation period. The sequence of eugenin has been determined using a combination of positive and negative ion electrospray mass spectrometry. This compound bears some structural resemblance to the mammalian neuropeptide cholecystokinin 8 [AspTyr(SO 3 )MetGlyTrpMetAspPhe-NH 2 ] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO 3 )ThrGlyTrpMetAspPhe-N H 2 ]. Eugenin has been synthesized by a route which causes only minor hydrolysis of the sulfate group when the peptide is removed from the resin support. Bio- logical activity tests with eugenin indicate that it contracts smooth muscle at a concentration of 10 )9 m, and enhances the proliferation of splenocytes at 10 )7 m, probably via activation of CCK 2 receptors. The activity of eugenin on splenocytes suggests that it is an immunomodulator peptide which plays a role in the protection of pouch young. Abbreviations CCK-8, cholecystokinin 8; CCK-8-NS, cholecystokinin 8 nonsulfated; QTOF, quadrupole-time-of-flight; splenocyte, spleen derived lymphocyte; TFA, trifluoroacetic acid. FEBS Journal 272 (2005) 433–443 ª 2005 FEBS 433 marsupials (including the Tammar wallaby) [8]. There is thus an apparent conflict due to the seemingly unprotected young crawling into, and subsequently developing in, a pouch abundant with harmful micro- organisms. There are three possible scenarios which may explain how the female wallaby protects the young during the early period of occupancy in the pouch. She may have antimicrobial and other biologically active agents in her milk, there may be host defence compounds in the secretion contained in the pouch, or there may be host defence compounds in the saliva, which she deposits when licking the pouch. It is known that (a) there are antimicrobial peptides in the pouch of the koala (Phascolarctos cinereus) [9], and (b) there are anti- microbially active proteins and peptides, including immunoglobulins, lysozyme and other antibacterial enzymes, in the milk of higher animals [7,10–18]. In this context, marsupial whey proteins have been exam- ined as a function of the time when they are present during the lactation period [19–25]; generally the pro- tein content varies significantly from the early to the late lactation period. Female wallabies produce a waxy secretion in the pouch, and the constituency of this secretion appears to depend upon the oestrus cycle and the time the young has spent in the pouch [6]. There is also evi- dence that polyprotodont opossums produce immuno- globulins in the pouch [26]. Whether immunoglobulins are secreted into the pouch of diprotodonts such as the Tammar wallaby is yet to be established. In this paper we report a study of the low molecular mass (< 2000 Da) water-soluble components of swabs taken from the pouch of the Tammar wallaby [27], with a view to identifying any maternal defence com- pounds (e.g. antimicrobial agents and ⁄ or neuropep- tides) in the pouch. We describe a unique mammalian cholecystokinin (CCK)-like peptide, eugenin, which may act as an immunomodulator. Results The pouch swabs of female wallabies with or without young in the pouch, contain low molecular mass (< 2000 Da) water-soluble compounds. Figure 2 shows a typical HPLC separation. MS and MS ⁄ MS data on the components of all HPLC fractions indicate the presence of a variety of lipid, sugar and phosphate Fig. 1. The young of Macropus eugenii (A) climbing into the pouch and (B) attached to a teat. Fig. 2. HPLC of aqueous extract of pouch secretion from female Macropus eugenii carrying young during the early lactation period. Peak marked with an asterisk contains eugenin. Protection by eugenin of Macropus eugenii young R. V. Baudinette et al. 434 FEBS Journal 272 (2005) 433–443 ª 2005 FEBS containing systems which have not been fully character- ized. None of these fractions show antimicrobial activity at MIC values below 100 lgÆmL )1 , and with the excep- tion of one component, they have not been studied fur- ther. The exception is the only peptide identified (by MS ⁄ MS data) from the pouch swabs. This peptide was isolated in lg amounts from pouch swabs taken from early lactating females in the first two weeks of the occu- pancy of young in the pouch. We have called this peptide eugenin. Eugenin was not detected, following exhaustive monitoring, in pouch swabs from female Tammar wallabies that were either (a) not carrying young, or (b) were bearing young, but after the early lactation period (i.e. after the young had been resident in the pouch for more than two weeks). Monitored HPLC profiles of pouch swabs not containing eugenin were almost identi- cal with that shown in Fig. 2, except that the fraction corresponding to that designated with an asterisk (Fig. 2), is reduced in abundance to the baseline. Structure determination of eugenin Because eugenin has an N-terminal pGlu residue, automated Edman sequencing [28] cannot be used to determine the amino acid sequence of this peptide. Sequence analysis was effected using positive and neg- ative ion electrospray mass spectrometry. The negative ion mass spectrum of eugenin gives peaks corresponding to (M-H) – and [(M-H) – -SO 3 ] – at m ⁄ z 1371 and 1291, respectively, indicating that euge- nin has a molecular mass of 1372 Da, and that it con- tains a sulfate group. The positive ion mass spectrum shows a small MH + ion at m ⁄ z 1373, and a pro- nounced peak corresponding to an [MH + -SO 3 ] + spe- cies at m ⁄ z 1293. The collision induced mass spectrum (MS ⁄ MS) of the [MH + -SO 3 ] + ion is recorded in Fig. 3. A partial amino acid sequence for eugenin was determined using B and Y+2 fragmentations (positive ion fragmentations of peptides reviewed in [29]). The B fragmentations are indicated schematically above the spectrum and provide information concerning the sequence from the C-terminal end of the peptide, while the Y+2 fragmentations (shown schematically under- neath the spectrum) provide sequencing data from the N-terminal end of the peptide. The positive ion mass spectrum (Fig. 3) provides the majority of the sequence except that it does not identify the first two residues at the N-terminal end of the peptide. Fig. 3. Positive ion mass spectrum (MS ⁄ MS) of the [MH + –SO 3 ] + ion of eugenin. B and Y+2 fragmentation sequences are indicated schema- tically above and below the spectrum, respectively. (Positive ion cleavages of peptides discussed in [29]). Figure scaled as follows: m ⁄ z 1286–1042 (·15), 1042–994 (·5), 994–772 (·15), 759–624 (·10), 317–175 (·5). Micromass QTOF2 instrument. R. V. Baudinette et al. Protection by eugenin of Macropus eugenii young FEBS Journal 272 (2005) 433–443 ª 2005 FEBS 435 The collision induced negative ion mass spectrum (MS ⁄ MS) of the [(M-H) – -SO 3 ] – ion of eugenin is shown in Fig. 4. There are a number of backbone cleavages in negative ion spectra which provide sequencing information. These have been described previously [30]. Two of these (a and b cleavages) are fragmentations of amide moieties, and give infor- mation analogous to that provided by B and Y+2 cleavages in the corresponding positive ion spectra. The other backbone cleavages (d and c processes) ori- ginate from Asp, Asn, Glu or Gln side chains and provide specific information concerning the positions of these four residues. The d and c fragmentations are particularly important in identifying Gln residues, because isobaric Gln and Lys cannot be differentiated by low resolution positive ion mass spectrometry. The a and b derived sequences are indicated schematically above and below the negative ion spectrum shown in Fig. 4, while d and c cleavages are indicated on the spectrum. The data shown in Fig. 4 gives the sequence of eugenin except that it does not indicate the relative orientation of residues 6 and 7. The spec- trum identifies pGlu as residue 1 and shows that resi- due 2 is Gln rather than Lys. A combination of the fragmentation data from the negative and positive ion spectra give the full sequence of eugenin (for sequence, see Table 1). Synthesis of eugenin Eugenin was synthesized to confirm the structure of the compound, and to provide sufficient material to allow biological testing to be performed. The synthesis of tyrosine sulfate containing peptides can be challenging because of possible hydrolysis of the sulfate residue occurring during synthesis, in Table 1. Eugenin, and mammalian and amphibian analogues. Sequence Name pGluGlnAspTyr(SO 3 )ValPheMetHis- ProPhe-NH 2 Eugenin AspTyr(SO 3 )MetGlyTrpMetAspPhe-NH 2 Cholecystokinin-8 [37] Tyr(SO 3 )GlyTrpMetAspPhe-NH 2 Hexagastrin [38] pGluGlnAspTyr(SO 3 )ThrGlyTrpMetAsp- Phe-NH 2 Caerulein [39,40] pGluGlnAspTyr(SO 3 )ThrGlyTrpPheAsp- Phe-NH 2 Caerulein 1.2 [41] pGluAsnAspTyr(SO 3 )LeuGlyTrpMetAsp- Phe-NH 2 D 2 L 5 -Caerulein [58] pGluGluTyr(SO 3 )ThrGlyTrpMetAspPhe-NH 2 Phyllocaerulein [59] Fig. 4. Negative ion mass spectrum of the [(M-H) – -SO 3 ] – ion of eugenin. a and b fragmentation sequences are drawn schematically above and below the spectrum, respectively. d and c cleavages are shown on the spectrum. (Backbone cleavage ions in negative ion spectra discussed in [30]). Figure scaled as follows: m ⁄ z 1284–1044 (·80), 1012–561 (·10), 560–248 (·5), 247–52 (·50). Micromass QTOF2 instrument. Protection by eugenin of Macropus eugenii young R. V. Baudinette et al. 436 FEBS Journal 272 (2005) 433–443 ª 2005 FEBS particular when the synthesized peptide is removed from the resin support. It has been reported that the peptide-resin cleavage and the removal of protecting groups can be effected using trifluoroacetic acid (TFA) at low temperature with only minimal damage to the Tyr(SO 3 ) residue [31,32]. The procedure used for the synthesis of eugenin is a modification of the reported methods, and is outlined in detail in Experimental pro- cedures. The key step involves treating the peptide- resin with TFA ⁄ tri-isopropyl silane (9 : 1) at 4 °C for 2.5 h under nitrogen, a method which releases the deprotected peptide from the resin with only minor hydrolysis of the Tyr(SO 3 ) residue. Preparative HPLC of the reaction product gives analytically pure eugenin, MH + ¼ 1373 Da. Synthetic and natural eugenin were shown to be identical by negative and positive ion mass spectrometry (MS and MS ⁄ MS) and HPLC. Biological testing As eugenin had similar structural elements to both CCK and caerulein, known CCK receptor agonists, we performed biological activity screening in tissues with well-characterized CCK responses. Contraction studies Acetylcholine contracted guinea pig ileal segments in a concentration-dependent fashion (data not shown). The mixed CCK 1 CCK 2 receptor agonist and standard CCK-8 produced potent increases in contraction, was maximally effective at 10 )9 m, but produced only about 60% of the contraction produced by the maximally effective concentration of acetylcholine (Fig. 5A). The CCK 2 agonist and standard cholecy- stokinin 8 nonsulfated (CCK-8-NS) also produced increases in contraction, but was less potent and less effective than CCK-8 (Fig. 5A). These results are con- sistent with previous studies [33]. Eugenin also pro- duced an increase in contraction, and was equieffective and equipotent with CCK-8-NS (Fig. 5A). This sug- gested that eugenin might be acting as a CCK 2 agon- ist. As the contraction produced by CCK 2 agonists is due to the release of acetylcholine from cholinergic nerve terminals, the effects of eugenin and CCK-8 were determined in the presence of atropine (10 )6 m). This concentration of atropine was sufficient to com- pletely block the effects of the maximally effective con- centration of acetylcholine (data not shown). Atropine had no effect on the contraction produced by CCK-8. However atropine completely stopped the contraction produced by 10 )8 m eugenin and substantially reduced the contraction produced by 10 )7 m eugenin (Fig. 5B). Spleen derived lymphocyte proliferation studies The result that eugenin is a CCK 2 agonist has import- ant implications for maternal defense of the pouch young. Lymphocytes have CCK 2 receptors, which when stimulated, result in proliferation. Spleen derived lymphocyte (splenocyte) proliferation was assessed using the Alamar Blue fluorescence dye method [34]. CCK-8 produced a concentration dependent increase in lymphocyte proliferation in both the presence (Fig. 6A) and absence (data not shown) of the mito- gen concanavalin A. CCK-8-NS was less effective (Fig. 6A). This is consistent with previous studies [35,36]. Eugenin (and to a lesser extent, desulfated eugenin) also produced a concentration dependent increase in lymphocyte proliferation in both the pres- Fig. 5. (A) CCK-8 (d), CCK-8-NS (h) and eugenin ( ) concentration– response curves in guinea pig ileum. Ileal segments were exposed to increasing concentrations of CCK-8, CCK-8-NS and eugenin. Con- tractions were measured on a Maclab data recorder (Maclab, Castle Hill, New South Wales, Australia) and expressed as a percentage of the maximal acetylcholine response (10 )6 M; 56 ± 15 mm). Data are expressed as mean ± SD of three independent experiments. (B) The effect of atropine on contractions produced by CCK-8 and eugenin in guinea pig ileum. Ileal segments were exposed to either vehicle or atropine (10 )6 M) for 15 min then CCK-8 or eugenin applied. Contractions were measured on a Maclab data recorder and expressed as a percentage of the maximal acetylcholine response (10 )6 M; 86 ± 15 mm). Data are expressed as mean ± SD of two experiments, except for eugenin vehicle, where n ¼ 1. R. V. Baudinette et al. Protection by eugenin of Macropus eugenii young FEBS Journal 272 (2005) 433–443 ª 2005 FEBS 437 ence (Fig. 6B) and absence (data not shown) of conca- navalin A. Discussion Eugenin is the only peptide detected in aqueous extracts of pouch swabs of the Tammar wallaby. Euge- nin has a sequence related to those of the mammalian gastrin-like neuropeptides CCK-8 [37] and hexagastrin [38] (Table 1). Eugenin also shows significant similarity to the amphibian caerulein neuropeptides [39–41]. CCK-8 and caerulein have similar physiological activ- ity; they both show potent smooth muscle contraction, gastrin-like activity and they reduce blood pressure at concentrations as low as ngÆkg )1 of body weight. Caerulein is an analgesic several thousand times more potent than morphine [40]. CCK-8 and caerulein both contain a tyrosine sulfate residue; the bioactivity is diminished if the tyrosine sulfate group is hydrolysed [40]. Eugenin corresponds to the caeruleins in having the same first four residues, but the sequence after the Tyr(SO 3 ) residue of eugenin is different from those of the other mammalian and amphibian analogues shown in Table 1. CCK-8 and caerulein bind to CCK receptors [42]. There are two types of CCK receptor, CCK 1 and CCK 2 , differing in anatomical locations and actions [43]. The sequences of the CCK receptors are known [44] and representations of their 3D structures have been reported [44,45]. Both NMR and other experi- mental data have been used to determine where CCK- 8 binds on the receptors [44–47]. In the present study we use CCK-8 and its desulfated analogue (CCK-8- NS) as standards. CCK-8 and caerulein activate both CCK receptors: perhaps eugenin may act via one or both CCK receptor subtypes. In the guinea pig ileum, CCK receptor agon- ists act to cause contraction of smooth muscle [33]. CCK 1 receptors are present on the smooth muscle, and contract the smooth muscle directly. In contrast, CCK 2 receptors act indirectly, by causing the release of acetyl- choline from cholinergic nerves in the myenteric plexus, which activates muscarinic receptors on smooth muscle [33]. In the present study, the standard neuropeptide CCK-8, which activates CCK 1 and CCK 2 receptors, produced a concentration dependent increase in contraction of guinea pig isolated ileal segments. CCK- 8-NS, which is selective for CCK 2 receptors, also pro- duced concentration dependent contraction of ileal segments but was less potent and effective than CCK-8. These results are consistent with the results of Patel et al. [33]. Eugenin produced a concentration depend- ent contraction of ileal smooth muscle segments, with a similar potency to that of CCK-8-NS. To determine if eugenin acts through CCK 2 recep- tors, the effect of the muscarinic blocker atropine was investigated. Atropine had no effect on the response of CCK-8, but substantially reduced the response to euge- nin, indicating that eugenin is indeed acting through CCK 2 receptors. To further investigate the interaction of eugenin with CCK 2 receptors, we investigated the effect of eugenin on lymphocyte proliferation. Lymphoid cells have CCK 2 receptors exclusively [43,48], and exposure of lymphoid tumour cell lines [35] or mouse lymphocytes [36] to CCK agonists results in lymphocyte prolifer- Fig. 6. (A) CCK-8 (d) and CCK-8-NS (s) concentration–response curves in mouse splenocytes. Splenocytes were exposed to increasing concentrations of CCK-8, or a single concentration of CCK-8-NS in the presence of the mitogen concanavalin A. Lympho- cyte proliferation was measured by the increase in fluorescence due to conversion of Alamar Blue [37]. Data shown are from a sin- gle experiment performed in quadruplicate, representative of two experiments carried out in quadruplicate. (B) Eugenin ( ) and euge- nin-NS (h) concentration-response curves in mouse lymphocytes. Lymphocytes were exposed to increasing concentrations of euge- nin in the presence of the mitogen concanavalin A. Lymphocyte proliferation was measured by the increase in fluorescence due to conversion of Alamar Blue. Data were expressed as a percentage of the CCK maximum response (10 )5 M, performed in the same time period with each run). Values are the mean ± SD of four experiments for eugenin. Protection by eugenin of Macropus eugenii young R. V. Baudinette et al. 438 FEBS Journal 272 (2005) 433–443 ª 2005 FEBS ation. In these experiments, exposure of mouse spleno- cytes to the standard neuropeptide CCK-8 resulted in a concentration dependent increase in proliferation, as measured by the Alamar Blue assay [34]. These results are consistent with previous studies [35,36] (although Medina et al. [36] found CCK-8 to be more potent than in the current study, the cells were exposed to CCK agonists for 72 h compared to 24 h in this study). CCK-8-NS produced proliferation, but was less potent than CCK-8. Eugenin also produced an increase in proliferation, equieffective with CCK-8, while desulfated eugenin shows a much reduced response (Fig. 6B). These results are consistent with eugenin being a CCK 2 agonist. These results provide insight concerning the possible role of eugenin in the wallaby pouch. Eugenin is only observed during the early lactation period (i.e. when the young has no immune system of its own), when there is a profound fall in the microbial flora of the pouch [6]. Neither eugenin nor other low molecular mass components of the pouch have antibacterial properties per se. However the skin is also an active immune tissue, and as CCK 2 receptors have a role in stimulating immune cells [35,36,49], eugenin may act to stimulate the immune cells in the skin. As well as stimulation of the proliferation of lymphocytes, activa- tion of CCK receptors stimulate production of inter- leukins and secretion of immunoglobulins on the mucosal surface of the intestine [49,50]. The antibacte- rial defence of the intestinal mucosa depends in part on stimulation of CCK receptors [49]. From a consideration of the experimental data, we suggest that eugenin stimulates immune cells in the pouch of the Tammar wallaby in the early lactation per- iod, thus reducing bacterial flora numbers in the pouch. Experimental procedures Pouch swabs Cotton wool swabs of the pouches of three female M. euge- nii were taken at two day intervals, from two days before the young occupies the pouch until the pouch had been occupied for two weeks, and then weekly for the next four weeks. Each swab was shaken with deionized water (50 mL), the mixture diluted with an equal volume of meth- anol, centrifuged, filtered through a Millex HV filter unit (0.45 lm), and lyophilized (the methanol was added to denature and precipitate any enzymes which may effect degradation of active pouch components). This procedure provided, on average, 1–2 mg of solid material from each swab. Swabs were also taken, for comparison, from pouches of female M. eugenii that were not bearing young. HPLC separation of pouch material HPLC separation of pouch material was achieved using a VYDAC C18 HPLC column (5l, 300A, 4.6 · 250 mm) (Separations Group, Hesperia, CA, USA) equilibrated with 10% acetonitrile ⁄ aqueous 0.1% TFA. The lyophilized mix- ture (generally  1 mg) was dissolved in deionized water (50 lL), of which a 10 lL fraction was injected into the column. The elution profile was generated using a linear gradient produced by an ICI DP 800 Data Station control- ling two LC1100 HPLC pumps, increasing from 10 to 75% (v ⁄ v) acetonitrile over a period of 60 min at a flow rate of 1mLÆmin )1 . The eluant was monitored by ultraviolet absorbance at 214 nm using an ICI LC-1200 variable wave- length detector (ICI Australia, Melbourne, Australia). An HPLC trace is shown in Fig. 2. All fractions of all HPLC traces were monitored using positive ion electrospray mass spectrometry. MS and MS ⁄ MS data were obtained for all components of all HPLC fractions (see below for details of MS procedures). Eugenin was isolated from HPLC traces of animals in the first two weeks of lactation. The fraction containing eugenin is indicated by an asterisk in Fig. 2. Two further HPLC separations of the initial eugenin frac- tion (10–75% acetonitrile over a period of 60 min at a flow rate of 1 mLÆmin )1 .) were required in order to obtain a pure eugenin. The eugenin fraction was collected, concen- trated and dried in vacuo providing 15 lg of pure eugenin. Electrospray mass spectrometry Positive and negative electrospray mass spectra were meas- ured with a Micromass QTOF2 orthogonal acceleration quadrupole-time-of-flight mass spectrometer (Micromass, Manchester, UK) with a mass range to 10 000 Da. The QTOF2 is fitted with an electrospray source in an ortho- gonal configuration with the ZSPRAY interface. Samples were dissolved in acetonitrile ⁄ water (1 : 1, v ⁄ v) and infused into the electrospray source with a flow rate of 5 lLÆmin )1 . Conditions were as follows: capillary voltage 2.43 kV, source temperature 80 °C, desolvation temperature 150 °C and cone voltage 50–100 V. MS ⁄ MS data were acquired with the argon collision gas energy set to 50eV to give opti- mal fragmentation. Preparation of synthetic eugenin [pGluGlnAsp- Tyr(SO 3 )ValPheMetHisProPhe-NH 2 ] Materials Manual syntheses were performed with Fmoc-amino acids purchased from Bachem, Novabiochem and Aspen (Aspen, CO, USA). The Ramage Amide tricyclic linker was pur- chased from Bachem. Diisopropylcarbodiimide was from Aldrich (Castle Hill, New South Wales, Australia) and 2-(1H-benzotriazol-1-yl)-1 ,1,3,3-tetramethyl-uroniumhexafluoro- R. V. Baudinette et al. Protection by eugenin of Macropus eugenii young FEBS Journal 272 (2005) 433–443 ª 2005 FEBS 439 phosphate was obtained from Richelieu Biotechno- logies (Quebec City, Quebec, Canada). N,N-diisopropyl- ethylamine, N,N-dimethylformamide, dichloromethane, piperidine, TFA and Fmoc-sulfotyrosine (all peptide syn- thesis grade) were purchased from Auspep (Melbourne, Australia). Acetone (HPLC grade) was obtained from Water Millipore (Milford, MA, USA). High purity water was generated by a Milli-Q TM purification system (Milli- pore, Bedford, MA, USA). Screw-cap glass peptide synthe- sis reaction vessels (20 mL) with a #2 sintered glass filter frit and a shaker for manual solid-phase synthesis were obtained from Embel Scientific Glassware (Brisbane, Queensland, Australia). Protocol and chain assembly The solid-phase peptide synthesis of eugenin was conducted manually on a 0.25 mmol scale by a standard method which has been reported earlier [51,52]. The determination of residual free a-amino groups following each cycle was monitored by the quantitative Ninhydrin test [53], except for couplings to proline where a coupling efficiency of > 99.5% was achieved as shown by Isatin tests [54,55]. Deprotection and removal from resin The peptide-resin (337 mg) was treated with TFA (61.2 mL) in triisopropylsilane (6.8 mL) (9 : 1, v ⁄ v) at 4 °C for 2.5 h. The resin was removed and the TFA solution was concentrated under nitrogen. The crude peptide was washed with diethyl ether (10 mL), dissolved in aqueous acetonitrile [50%, 20 mL, containing 0.1% (v ⁄ v) TFA] and lyophilized to give a white powder (18 mg) [31,32]. HPLC analysis The peptide mixture (9 mg) was purified by preparative HPLC using a Vydac C18 column (10 lm, 2.2 · 25 cm). Chromatographic separations were achieved using linear gra- dients of solvent B in A at a flow of 8 mLÆmin )1 with 25–45% B over 40 min: solvent A, 100% water, 0.05% (v ⁄ v) TFA; solvent B, 90% (v ⁄ v) aqueous acetonitrile, 0.043% (v ⁄ v) TFA. The eluant was monitored at 230 nm. Lyophiliza- tion of the separated fractions gave eugenin (6 mg) [identical in HPLC retention time and mass spectra (both negative and positive ion) with natural eugenin] and desulfated eugenin (2 mg) (pGluGlnAspTyrValPheMetHisProPhe-NH 2 ). Bioactivity assays Antimicrobial testing on HPLC fractions and synthetic euge- nin was carried out by the Microbiology Department of the Institute of Medical and Veterinary Science (Adelaide, Australia) using a standard procedure [56]. The microorgan- isms used were: Bacillus cereus, Escherichia coli, Leuconostoc lactis, Listeria innocua, Micrococcus luteus, Pasteurella multo- cida, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus uberis. Neither the HPLC fractions nor euge- nin showed activity at an MIC value of 100 lg Æ mL )1 against any of these organisms, and is thus deemed inactive. Contraction studies Drugs and materials This work was approved by The University of Adelaide Animal Ethics Committee. Acetylcholine, atropine, concanavalin A, CCK-8 and CCK-8-NS were obtained from Sigma-Aldrich. Alamar blue was obtained from Astral Scientific (Caringbar, New South Wales, Australia). Guinea pigs weighing approximately 300 g were used. Immediately before the experiment, the guinea pigs were killed by stunning and subsequent decapitation. The ileum was dissected free and was cleansed by rinsing with physiolo- gical salt solution (composition in mm): KCl 2.7, CaCl 2 1.0, NaHCO 3 13.0, NaH 2 PO4 3.2, NaCl 137, glucose 5.5 (pH 7.4), and mesenteric tissue was removed. Segments of about 3 cm were cut, which were suspended in 20 mL organ baths containing the physiological salt solution and were gassed with 95% O 2 and 5% CO 2 . Segments were connected to a tissue holder and to an isometric force-displacement transducer. Tension was recorded via maclab v 3.0. Seg- ments were washed thoroughly by replacing the physiological salt solution repeatedly, and were then allowed to equilibrate for a period of 30 min under 2 g of resting tension. Supply reservoirs and organ baths were maintained at 37 °C and were gassed with O 2 ⁄ CO 2 as outlined above. Following the 30 min equilibration period, the tissue- bathing solution was replaced repeatedly with fresh drug- free physiological salt solution until a stable baseline tension was achieved. The tension was then readjusted to 2 g. All segment preparations were then constricted with acetylcholine (0.01–1 lm). After washout, acetylcholine (1 lm) was used again to check that the response was sta- ble. After 5 min washout and achievement of a stable base- line, a cumulative response curve to CCK-8 (10 -10 )10 -8 m) was performed. After another 5 min washout and achieve- ment of a stable baseline, a cumulative concentration response curve to either eugenin (10 -9 )10 -7 m) or CCK-8- NS (10 -9 )10 -7 m) was performed. In some experiments, fol- lowing washout, tissues were either pretreated with atropine or vehicle and CCK-8 or eugenin reapplied. Splenocyte proliferation studies Male Balb ⁄ C mice aged 6–8 weeks were used. Lymphocytes were prepared as described previously [57] with minor modifications. Aseptic techniques were used during Protection by eugenin of Macropus eugenii young R. V. Baudinette et al. 440 FEBS Journal 272 (2005) 433–443 ª 2005 FEBS preparation of the lymphocytes. Mice were killed by cervi- cal dislocation followed by prompt removal of the spleen. The spleen was prepared as a single-cell suspension by mas- saging and washing through a nylon mesh into a 15 mL tube with up to 15 mL of RPMI 1640 (Hepes modification, 0.3 mgÆmL )1 of l-glutamine and 5 mL of penicillin ⁄ strepto- mycin solution per litre). The cells were centrifuged at 4 °C for 5 min at 100 g, the supernatant material discarded and the cells resuspended in 1 mL of media followed by the addition of 10 mL of ice-cold lysis buffer (1 mL of 20.56 gÆL )1 tris base (pH 7.65), 9 mL of 0.83% NH 4 Cl in water, mixed just prior to addition to cells). The suspension was placed on ice for 4 min, centrifuged (5 min at 100 g) and the supernatant material discarded. The suspensions of cells were pooled and were resuspended in 10 mL of media followed by centrifugation (5 min at 100 g), removal of supernatant material and resuspended in 5 mL of enriched RPMI 1640 (RPMI 1640 enriched with 10% fetal bovine serum). The number of viable lymphocytes in the suspen- sion was counted using trypan blue and a haemocytometer. Cells were then diluted in enriched media to 1 · 10 6 cellsÆ mL )1 and 100 lL of this suspension was added to each well of the 96 multiwell plates (TTP, Zurich, Switzerland) to give a final volume of 200 lL, and final cell count of 50 000 cells per well. Either vehicle or the mitogen concanavalin 1 (2.5 lgÆmL )1 final concentration) was added to the wells, and then 10 lL of RPMI 1640 medium containing either CCK-8, CCK-8-NS or eugenin (to produce final concentrations of 10 -7 )10 -5 m) was added to the plate. Plates were incubated at 37 °C, using 5% CO 2 in a humidified incubator (Thermoline, Sydney, New South Wales, Australia) for 24 h. Twenty-five microlit- ers of the mitochondrial activity indicator dye Alamar Blue [34] was then added to give a final concentration of 2.5 lgÆmL )1 , and the plates incubated as above for a further 4 h. 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The bacterial content of the pouch diminishes significantly upon arrival and occupancy of the young marsupial [6]. When the young first crawls into the pouch,