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 en
Trang 1An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii
Russell V Baudinette1,*, Pinmanee Boontheung2, Ian F Musgrave3, Paul A Wabnitz2,
Vita M Maselli2, Jayne Skinner1, Paul F Alewood4, Craig S Brinkworth2and John H Bowie2
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(SO3)ValPheMetHisProPhe-NH2] 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(SO3)MetGlyTrpMetAspPhe-NH2] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO3)ThrGlyTrpMetAspPhe-NH2] 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)9m, and enhances the proliferation of splenocytes at
10)7m, probably via activation of CCK2 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.
Trang 2marsupials (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 immunoimmuno-globulins
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.
Trang 3containing 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)–-SO3]– 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+-SO3]+ spe-cies at m⁄ z 1293 The collision induced mass spectrum (MS⁄ MS) of the [MH+-SO3]+ 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 + –SO3] + 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.
Trang 4The collision induced negative ion mass spectrum
(MS⁄ MS) of the [(M-H)–-SO3]– 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.
pGluGlnAspTyr(SO 3 )ValPheMetHis-ProPhe-NH 2
Eugenin
AspTyr(SO3)MetGlyTrpMetAspPhe-NH2 Cholecystokinin-8 [37] Tyr(SO3)GlyTrpMetAspPhe-NH2 Hexagastrin [38] pGluGlnAspTyr(SO 3
)ThrGlyTrpMetAsp-Phe-NH2
Caerulein [39,40]
pGluGlnAspTyr(SO3 )ThrGlyTrpPheAsp-Phe-NH 2
Caerulein 1.2 [41]
pGluAsnAspTyr(SO3 )LeuGlyTrpMetAsp-Phe-NH2
D2L5-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.
Trang 5particular 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(SO3) 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(SO3) 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 CCK1 CCK2receptor agonist and standard
CCK-8 produced potent increases in contraction, was
maximally effective at 10)9m, but produced only
about 60% of the contraction produced by the
maximally effective concentration of acetylcholine
(Fig 5A) The CCK2 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 CCK2
agon-ist As the contraction produced by CCK2 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)6m)
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)8m eugenin and substantially reduced
the contraction produced by 10)7meugenin (Fig 5B)
Spleen derived lymphocyte proliferation studies The result that eugenin is a CCK2 agonist has import-ant implications for maternal defense of the pouch young Lymphocytes have CCK2 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)6M ; 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)6M ) 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)6M ; 86 ± 15 mm) Data are expressed as mean ± SD
of two experiments, except for eugenin vehicle, where n ¼ 1.
Trang 6ence (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(SO3) 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, CCK1 and CCK2, 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] CCK1receptors are present on the smooth muscle, and contract the smooth muscle directly In contrast, CCK2 receptors act indirectly, by causing the release of acetyl-choline from acetyl-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 CCK1 and CCK2 receptors, produced a concentration dependent increase in contraction of guinea pig isolated ileal segments CCK-8-NS, which is selective for CCK2 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 segmdepend-ents, with a similar potency to that of CCK-8-NS
To determine if eugenin acts through CCK2 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 CCK2receptors
To further investigate the interaction of eugenin with CCK2 receptors, we investigated the effect of eugenin
on lymphocyte proliferation Lymphoid cells have CCK2 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)5M , performed in the same
time period with each run) Values are the mean ± SD of four
experiments for eugenin.
Trang 7ation 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 CCK2agonist
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 CCK2 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 producactiva-tion 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-niiwere 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
1 mLÆmin)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 80C, 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(SO3)ValPheMetHisProPhe-NH2]
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
Trang 82-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluoro-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-QTM 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 fracLyophiliza-tions 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-NH2)
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 epidermidisand Streptococcus uberis Neither the HPLC fractions nor euge-nin showed activity at an MIC value of 100 lgÆmL)1against 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, CaCl21.0, NaHCO3 13.0, NaH2PO4 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% O2and 5% CO2 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 37C and were gassed with O2⁄ CO2as 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)10-8m
) was performed After another 5 min washout and achieve-ment of a stable baseline, a cumulative concentration response curve to either eugenin (10-9)10-7m
) or
CCK-8-NS (10-9)10-7m
) 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
Trang 9preparation 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)1of l-glutamine and 5 mL of penicillin⁄
strepto-mycin solution per litre) The cells were centrifuged at 4C
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% NH4Cl 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· 106 cellsÆ
mL)1and 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)10-5m
) was added to the plate Plates were incubated at 37C, using
5% CO2 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 After this, 175 lL aliquots were pipetted from each well
into a white 96 well plate, and fluorescence measured in a
Polestar Galaxy (BMG Labtechnologies, Durham, NC,
USA) fluorescent plate reader (excitation 544 nm, emission
590 nm)
Acknowledgements
We thank the Australian Research Council for
provi-ding maintenance funprovi-ding for this project The ARC
also provided the following stipends: C.S.B (research
associate), V.M.M and P.A.W (postgraduate
scholar-ships)
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