logy 561 (2007) 32–38www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Aged garlic extract and S-allyl cysteine prevent formationof advanced glycation endproducts

Muhammad Saeed Ahmad a, Monika Pischetsrieder b, Nessar Ahmed a,⁎

a School of Biology, Chemistry and Health Science, Manchester Metropolitan University, Oxford Road, Manchester M1 5GD, United Kingdomb Institute of Pharmacy and Food Chemistry, Friedrich Alexander Universitat, Erlangen-Nurnberg, Schuhstr. 19, D-91052 Erlangen, Germany

Received 23 October 2006; received in revised form 9 January 2007; accepted 11 January 2007Available online 1 February 2007

Abstract

Hyperglycaemia causes increased protein glycation and the formation of advanced glycation endproducts which underlie the complications ofdiabetes and ageing. Glycation is accompanied by metal-catalysed oxidation of glucose and Amadori products to form free radicals capable ofprotein fragmentation. Aged garlic extract is a potent antioxidant with established lipid-lowering effects attributed largely to a key ingredientcalled S-allyl cysteine. This study investigated the ability of aged garlic extract and S-allyl cysteine to inhibit advanced glycation in vitro. Bovineserum albumin (BSA) was glycated in the presence of Cu2+ ions and different concentrations of aged garlic extract and protein fragmentationwas examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Lysozyme was glycated by glucose ormethylglyoxal in the presence of different concentrations of aged garlic extract or S-allyl cysteine with subsequent analysis of glycation-derived crosslinking using SDS-PAGE. Amadori-rich protein was prepared by dialysing lysozyme that had been glycated by ribose for 24 h.This ribated lysozyme was reincubated and the effects of aged garlic extract, S-allyl cysteine and pyridoxamine on glycation-inducedcrosslinking was monitored. Aged garlic extract inhibited metal-catalysed protein fragmentation. Both aged garlic extract and S-allyl cysteineinhibited formation of glucose and methylglyoxal derived advanced glycation endproducts and showed potent Amadorin activity whencompared to pyridoxamine. S-allyl cysteine inhibited formation of carboxymethyllysine (CML), a non-crosslinked advanced glycationendproduct derived from oxidative processes. Further studies are required to assess whether aged garlic extract and S-allyl cysteine can protectagainst the harmful effects of glycation and free radicals in diabetes and ageing.© 2007 Elsevier B.V. All rights reserved.

Keywords: Glycation; Aged garlic extract; S-allyl cysteine; Diabetes; Antioxidant

1. Introduction

Diabetes mellitus is a metabolic disorder characterized bychronic hyperglycaemia and hyperlipidaemia that predisposesaffected individuals to long-term micro- and macrovascularcomplications of which cardiovascular disease is the mostserious consequence. During hyperglycaemia, body proteinsundergo increased glycation where glucose reacts non-enzy-matically with protein amino groups to form a labile Shiff basethat rearranges to a stable Amadori product. This Amadoriproduct undergoes further reactions involving dicarbonylintermediates such as 3-deoxyglucosone and methylglyoxal to

⁎ Corresponding author. Tel.: +44 161 247 1163; fax: +44 161 247 6357.E-mail address: N.Ahmed@mmu.ac.uk (N. Ahmed).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.01.041

form complex, heterogenous, fluorescent and crosslinkedstructures called advanced glycation endproducts.

Accumulation of crosslinked advanced glycation endpro-ducts in body tissues are believed to be responsible for the long-term complications of diabetes and ageing (Ahmed, 2005).However, the major advanced glycation endproduct in vivo iscarboxymethyllysine (CML), which is not a crosslink butformed by oxidative breakdown of Amadori products (Reddyet al., 1995) and its level increases two-fold in the skin ofdiabetic patients (Dyer et al., 1993). In the presence of oxygenand transition metals, glucose can undergo autoxidation(autoxidative glycation) as can Amadori products (glycoxida-tion) to produce free radicals capable of damaging proteins,lipids and nucleic acids (Hunt et al., 1993). Indeed, diabetesmellitus and ageing are associated with a build up of tissueadvanced glycation endproducts, increased oxidative stress and

33M.S. Ahmad et al. / European Journal of Pharmacology 561 (2007) 32–38

a decline in antioxidant status (Maritim et al., 2003).Furthermore, circulating serum advanced glycation endproductscan interact with cellular receptors (termed RAGE) to activatenuclear factor-kappa B (NF-kB) which in turn generates pro-inflammatory molecules and oxidative stress (Stern et al., 2002).

The involvement of advanced glycation endproducts indiabetic complications and ageing has prompted a search forcompounds capable of inhibiting their formation (Rahbar andFigarola, 2003). Recent studies suggest that compounds withcombined anti-glycation and antioxidant properties offermaximum protection against glucose-induced cellular damage(Duraisamy et al., 2003).

Aminoguanidine, a nucleophilic hydrazine has received themost interest and inhibits advanced glycation endproductformation both in vitro and in vivo by reacting with carbonylgroups of reducing sugars, Amadori products and intermediatessuch as methylglyoxal and 3-deoxyglucosone (Thornalley,2003). Aminoguanidine can act as an antioxidant at high con-centrations but has been shown to inhibit catalase and promotethe formation of hydrogen peroxide and associated free radicals(Ou and Wolff, 1993). Phase III clinical trials investigating theprotective effects of aminoguanidine against diabetic nephrop-athy were terminated because of safety concerns as somepatients developed flu-like symptoms, gastrointestinal problemsand anaemia (Ahmed, 2005; Thornalley, 2003). Other com-pounds undergoing clinical trials include pyridoxamine andthiamine pyrophosphate both of which are “Amadorins” orcompounds that prevent conversion of Amadori products toadvanced glycation endproducts (Khalifah et al., 1999). Recentlythere has been interest in natural products with anti-glycationproperties, for example, rutin from tomato paste is a potentinhibitor of advanced glycation endproducts in vitro (Kiho et al.,2004). Polyphenolic compounds from Luobuma tea are known tobe more potent than aminoguanidine in inhibiting advancedglycation endproducts (Yokozawa and Nakagawa, 2004).

Garlic (Allium sativum) has been used historically formedicinal purposes, particularly for treatment of diseases asso-ciated with ageing (Rahman, 2003). Aged garlic extractcontains potent antioxidant activity and is prepared fromnatural garlic that is aged for 20-months reducing its harshirritating taste and odour. However, this aged garlic has a greaterconcentration of organosulphur compounds such as S-allylcysteine which is a potent antioxidant and free radical scavenger(Imai et al., 1994). Although numerous studies have demon-strated the antioxidant properties of aged garlic extract, itsability to inhibit formation of advanced glycation endproducts isunknown. In this study, we investigated the ability of agedgarlic extract to inhibit metal-catalysed protein fragmentationduring experimental glycation. We also investigated the abilityof aged garlic extract to inhibit formation of glucose- andmethylglyoxal derived crosslinked advanced glycation end-products in vitro. Since S-allylcysteine is a key component ofaged garlic extract, we investigated its ability to inhibit for-mation of glucose and methylglyoxal derived crosslinked ad-vanced glycation endproducts and CML in vitro. The Amadorinactivity of aged garlic extract and S-allyl cysteine wasinvestigated and compared to pyridoxamine.

2. Materials and methods

2.1. Materials

Kyolic® liquid aged garlic extract and S-allyl cysteine werekindly provided by Wakunaga Pharmaceutical Company,Tokyo, Japan. Bovine serum albumin (fraction V, essentiallyfatty acid free), lysozyme, pyridoxamine, and methylglyoxalwere purchased from Sigma-Aldrich Company, Poole, UK. Thestandard protein assay kit and acrylamide solution wereobtained from Bio-Rad Laboratories, Hemel Hempstead, UK.Sodium dodecyl sulphate was obtained from ICN BiomedicalIncorporation, Ohio, USA. Dialysis tubing with a molecularweight cut off of 3.5 kDa was obtained from Pierce ChemicalCompany, Northumberland, UK.

2.2. In vitro glycation of proteins

BSA or lysozyme (10 mg/ml) were incubated in either 0.5 Mglucose or 0.1 M methylglyoxal ±20 μM Cu (II) sulphate ±3–84 mg/ml aged garlic extract or 1–100 mM S-allyl cysteine in0.1 M sodium phosphate buffer containing 3 mM sodium azide,pH 7.4 at 37 °C for up to 35 days according to an establishedprocedure (Duraisamy et al., 2003). Samples were stored frozenat −20 °C before analysis.

2.3. Amadorin activity of aged garlic extract and S-allylcysteine

Amadorin activity was determined using a post-Amadoriscreening assay (Khalifah et al., 1999). Lysozyme (10 mg/ml)was incubated with 0.5 M ribose in 0.1 M sodium phosphatebuffer containing 3 mM sodium azide, pH 7.4 at 37 °C for 24 h.Unbound ribose was removed by dialysis against 4 l of 0.1 Msodium phosphate buffer, pH 7.4 at 4 °C for 48 h with 5–6changes. Following dialysis, the protein concentration wasdetermined using the Bio-Rad standard protein assay kit basedon the Bradford dye-binding procedure (Bradford, 1976).Dialysed ribated lysozyme (10 mg/ml) was reincubated ±3–56 mg/ml aged garlic extract or 5–50 mM pyridoxamine orS-allyl cysteine in 0.1 M sodium phosphate buffer containing3 mM sodium azide, pH 7.4 at 37 °C for 7 days.

2.4. Analysis of protein fragmentation and crosslinkedadvanced glycation endproducts

Glycation-induced protein fragmentation and crosslinkingwere assessed by sodium dodecyl polyacrylamide gel electro-phoresis (SDS-PAGE) using 10 or 15% polyacrylamide gels forBSA and lysozyme respectively followed by staining withCoomassie blue R (Laemmli, 1970). Protein samples werediluted 1 in 10 in 0.05 M Tris–HCl buffer, pH 6.8 containing1% (w/v) sodium dodecyl sulphate, 1% (v/v) 2-mercaptoethanoland 20% (v/v) glycerin and then boiled for 5 min. Samplescontaining 10 μg of protein were loaded into the wells followedby 2 μl of bromophenol blue and then subjected to elec-trophoresis using the mini-Protean® 3 apparatus (Bio-Rad

Fig. 1. (A) Gel showing BSA incubated for 21 days alone (lane a) or in thepresence 0.5 M glucose (lane b). BSA was also incubated in 0.5 M glucose,20 μM Cu2+ and 0, 14, 28, 42 and 56 mg/ml of aged garlic extract for 21 days(lanes c–g). (B) Image analysis of gel to show the percentage inhibition offragmentation in different concentrations of aged garlic extract.

Fig. 2. (A) Gel showing lysozyme incubated in the absence (lane a) or presenceof 0.5 M glucose (lanes b–i) for 35 days and the effect of 0, 7, 14, 28, 42, 56, 70and 84 mg/ml of aged garlic extract (lanes b–i) on dimerization. (B) Imageanalysis of gels to show the percentage inhibition of crosslinked advancedglycation endproduct formation in different concentrations of aged garlicextract.

34 M.S. Ahmad et al. / European Journal of Pharmacology 561 (2007) 32–38

Laboratories, Hemel Hempstead, UK). Gels were stained in asolution containing 0.25% (w/v) Coomassie blue R, 50% (v/v)methanol and 10% (v/v) acetic acid. Gels were destained in asolution containing 25% (v/v) methanol and 7% (v/v) aceticacid.

2.5. Image analysis of SDS-PAGE gels

The gels were photographed using a Fuji S2 Pro camera. Afree PC version of NIH Image, called Scion Image for Windowsavailable from Scion Corporation, was used for image analysis.Bands were compared within the same gel. Integrated Density(I.D.) was measured to analyze the one dimensional electro-phoretic gels and computed using the following formula:

I:D: ¼ N � ðmean−backgroundÞ:

Where N is number of pixels in the selection and thebackground is the modal grey value (most common pixel value)after smoothing the histogram. Sufficient background wasincluded in the selection to avoid errors.

Percentage inhibition of protein fragmentation: this wasassessed by measuring intensity of monomer bands for albuminusing the formula:

100−½100� ðI:D:without inhibitor−I:D:with inhibitorÞ=I:D:without inhibitor�:

Percentage inhibition of crosslinked advanced glycationendproducts: this was calculated using the following formula:

100� ðI:D:without inhibitor−I:D:with inhibitorÞ=I:D:without inhibitor:

2.6. Effect of S-allyl cysteine on formation of CML

CML proteins were synthesised and anti-CML antiserumwas prepared as described previously (Tauer et al., 1999). Anon-competitive ELISAwas performed as described previously(Humeny et al., 2002). Briefly, microtiter plates were coatedovernight at 4 °C with 100 μl of CML-lysozyme standards orsamples diluted 1:200 in 50 mM carbonate buffer (pH 9.7).Unless stated otherwise, the wells were washed twice after eachstep with PBS containing 0.05% Tween 20. The coated plateswere blocked by adding 200 μl of a 3% solution of skimmedmilk powder in double distilled water per well for 2 h at roomtemperature. This was followed by addition of 100 μl of CMLspecific antiserum which was diluted 1:20,000 in PBS contain-ing 0.2% BSA and 0.05% Tween 20 to each well and then theplates were shaken for 1 h at room temperature. After washingthe plates three times, 100 μl of peroxidase conjugated goatantirabbit IgG (diluted 1:5000 in 0.1% BSA in PBS) was addedto each well and the plates shaken for 1 h at room temperature.

Fig. 3. (A) Gel showing lysozyme incubated in the presence of 0.1 Mmethylglyoxal (lanes a–h) for 7 days and the effect of 0, 3, 14, 28, 42, 56, 70 and84 mg/ml of aged garlic extract (lanes b–h) on protein crosslinking. (B) Imageanalysis of gels to show the percentage inhibition of crosslinked advancedglycation endproducts in different concentrations of aged garlic extract.

35M.S. Ahmad et al. / European Journal of Pharmacology 561 (2007) 32–38

The wells were washed three times and the antibody bindingwas detected using 150 μl per well of tetramethylbenzidinediluted in substrate solution. This was prepared by dissolving345 μl of tetramethylbenzidine and 114 μl of 3% H2O2 in 20 mlof substrate buffer, the latter was prepared by dissolving 23.02 gKH2 citrate and 0.05 g K sorbate in 500 ml distilled water. Thereaction was stopped after 30 min by adding 50 μl per well of2 N sulphuric acid and the absorption was measured at 450 nmusing a microplate reader. Samples were analysed in duplicatesand the results are expressed as a percentage of those for CMLin the absence of S-allyl cysteine.

Fig. 4. Image analysis of gel to show the percentage inhibition of crosslinkedadvanced glycation endproducts following reincubation of ribated lysozymein different concentrations of pyridoxamine (5–50 mM) and aged garlic extract(3–56 mg/ml) for 7 days.

3. Results

3.1. Effect of aged garlic extract on protein fragmentation andcrosslinked advanced glycation endproducts

BSA incubated in the presence of glucose (Fig. 1A, lane b)produces a small amount of fragmentation not visible for BSAincubated alone (Fig. 1A, lane a). However, glycation of BSA inthe presence of Cu2+ ions generates free radicals that causeprotein fragmentation (Fig. 1A, lane c). Aged garlic extract at aconcentration of 56 mg/ml inhibits fragmentation by 74%(Fig. 1B). Glucose reacts slowly with lysozyme and a period of35 days was allowed for sufficient crosslinked advancedglycation endproducts to form. Crosslinking of lysozymecauses formation of dimers with an approximate molecularweight of 28.6 kDa (Fig. 2A). Aged garlic extract inhibitedcrosslinked advanced glycation endproducts causing a reduc-tion in intensity of the dimerised lysozyme band (Fig. 2A, lanesc–i). This inhibitory effect occurs in a dose-dependent mannerwith a maximum inhibition of 73% in samples containing84 mg/ml aged garlic extract (Fig. 2B). Methylglyoxal reactswith lysozyme much faster than glucose forming crosslinkedadvanced glycation endproducts after only 7 days of incubationto produce dimers, trimers and tetramers (Fig. 3A). The effect ofdifferent concentrations of aged garlic extract is best visible inthe trimer band which was used to calculate the percentageinhibition relative to lysozyme glycated in the absence of agedgarlic extract (Fig. 3B).

Reincubation of dialysed ribated lysozyme generated cross-linked advanced glycation endproducts that were inhibited inthe presence of increasing concentrations of pyridoxamine.There is a 55% reduction in glycation-derived crosslinking inthe presence of 50 mM pyridoxamine (Fig. 4). Aged garlicextract has Amadorin activity and inhibits crosslinked advancedglycation endproducts with maximal inhibition occurring in thepresence of 56 mg/ml aged garlic extract (Fig. 4).

3.2. Effect of S-allyl cysteine on crosslinked advancedglycation endproducts

S-allyl cysteine also inhibited formation of crosslinkedadvanced glycation endproducts in a dose-dependent manner

Fig. 5. Image analysis of gel to show the percentage inhibition of crosslinkedadvanced glycation endproducts following glycation of lysozyme by 0.5 Mglucose in different concentrations of S-allyl cysteine (10–80 mM) for 35 days.

Fig. 8. Effect of 0–80 mM S-allyl cysteine on formation of CML in lysozymeincubated with and without 0.5 M glucose for 35 days at 37 °C. CML formationis expressed in relation to the CML concentration of the lysozyme which wasincubated in the absence of S-allyl cysteine (results are expressed as mean±SD,n=3).

Fig. 6. Image analysis of gel to show the percentage inhibition of crosslinkedadvanced glycation endproducts following glycation of lysozyme by 0.1 Mmethylglyoxal in different concentrations of S-allyl cysteine (1–100 mM) for7 days.

36 M.S. Ahmad et al. / European Journal of Pharmacology 561 (2007) 32–38

with a maximum inhibition of 68% using an 80 mMconcentration of S-allyl cysteine (Fig. 5). Similarly, S-allylcysteine inhibited methylglyoxal derived crosslinked advancedglycation endproducts with a maximum inhibition of 66% insamples containing 100 mM S-allyl cysteine (Fig. 6).

S-allyl cysteine has Amadorin activity and inhibitedformation of crosslinked advanced glycation endproducts butwas not as effective as pyridoxamine at higher concentrations(Fig. 7).

All the above experiments on aged garlic extract and S-allylcysteine were repeated in three independent experiments andgave similar findings so only representative results are shown.

3.3. Effect of S-allyl cysteine on formation of CML

The results for CML are related to CML concentration oflysozyme which was glycated in the absence of S-allyl cysteine.S-allyl cysteine inhibited formation of CML in a dose-dependent manner (Fig. 8).

4. Discussion

Increased glycation during hyperglycaemia can cause intra-or inter molecular crosslinking of proteins as they accumulate

Fig. 7. Image analysis of gel to show the percentage inhibition of crosslinkedadvanced glycation endproducts following reincubation of ribated lysozyme indifferent concentrations (5–50 mM) of pyridoxamine and S-allyl cysteine for7 days.

advanced glycation endproducts. Numerous studies have shownthat build up of crosslinked advanced glycation endproducts onlong-lived proteins may underlie the development of complica-tions affecting diabetes and ageing (Ahmed, 2005; Baynes,2001). Furthermore, the levels of serum advanced glycationendproducts reflect the severity of these complications whereastherapeutic interventions aimed at reducing advanced glycationendproducts can inhibit or delay their progression (Ono et al.,1998; Monnier, 2003).

In recent years, the use of garlic as a health supplement hasreceived much interest particularly because of its beneficialeffects in protecting against coronary heart disease (Banerjeeand Maulik, 2002; Rahman, 2001). Such protective effects arelargely attributed to the antioxidant properties of garlic (Bane-rjee et al., 2003). In our study, aged garlic extract protectedagainst glycation-induced protein fragmentation probablybecause of its ability to chelate transition metals thereforepreventing autoxidative glycation and glycoxidation reactions.Indeed, previous work has shown that aged garlic extractchelates Cu2+ ions and protects against LDL oxidation (Dillonet al., 2003). Like other workers, this study has alsodemonstrated that exposure of proteins to glucose inducescrosslinking as they accumulate advanced glycation end-products (Prabhakaram and Ortwerth, 1994). Lysozyme is agood model protein for investigation of glycation-inducedcrosslinking in that oligomerisation occurs readily and isdetectable by SDS-PAGE. However, to our knowledge, this isthe first study demonstrating the ability of aged garlic extract toinhibit formation of crosslinked advanced glycation end-products in vitro. This inhibitory effect may be due to the factthat aged garlic extract can chelate transition metals as has beendemonstrated for a number of other substances (Price et al.,2001). Aged garlic extract may also inhibit advanced glycationendproducts because of its antioxidant properties as has beenreported for green tea (Rutter et al., 2003). Aged garlic extractinhibited formation of methylglyoxal derived advanced glyca-tion endproducts and may also act by blocking conversion ofdicarbonyl intermediates to advanced glycation endproducts.Furthermore, our results show that aged garlic extract possessesAmadorin activity. Exposure of lysozyme to ribose for a period

37M.S. Ahmad et al. / European Journal of Pharmacology 561 (2007) 32–38

of 24 h generates glycated protein rich in Amadori but notadvanced glycation adducts (Khalifah et al., 1999). Reincuba-tion of this ribated lysozyme in the absence of sugar generatescrosslinked advanced glycation endproducts as reportedpreviously (Liggins and Furth, 1996) but their formation isinhibited in the presence of aged garlic extract.

Aged garlic extract has more potent antioxidant capacitycompared to other garlic preparations because of highconcentrations of S-allyl cysteine but also S-allyl mercaptocys-teine, selenium and allixin, all of which are stable, bioavailableantioxidants (Imai et al., 1994; Borek, 2001). Our results showthat S-allyl cysteine inhibits glucose- and methylglyoxal derivedcrosslinked advanced glycation endproducts, CML and hasAmadorin activity. In addition to its antioxidant properties, theamino groups on S-allyl cysteine could react with carbonylgroups from reducing sugars, Amadori adducts and dicarbonylintermediates therefore blocking their conversion to advancedglycation endproducts. Dicarbonyl intermediates such asmethylglyoxal have received considerable attention asmediatorsof advanced glycation endproduct formation and are known toreact with lysine, arginine and cysteine residues in proteins toform glycosylamine protein crosslinks (Frye et al., 1998) whichare perhaps inhibited by both aged garlic extract and S-allylcysteine. Like pyridoxamine, both aged garlic extract and S-allylcysteine may inhibit at multiple stages of advanced glycationendproduct formation. This contrasts with other inhibitors, suchas aminoguanidine which has no Amadorin activity (Khalifah etal., 1999). Following absorption from the gastrointestinal tract,S-allyl cysteine is distributed largely in the plasma but also inthe liver and kidneys. Animal studies have shown that S-allylcysteine has very minor toxicity (Kodera et al., 2002). S-allylcysteine has been reported to inhibit NF-kB activation in a dose-dependent manner and may protect against intracellularoxidative stress which is generated following interactionbetween advanced glycation endproducts and RAGE (Ide andLau, 2001). The concentration of S-allyl cysteine is approxi-mately 1000 μg per gram of aged garlic extract and thiscompares to approximately 20 μg of S-allyl cysteine per gram ofraw garlic. Aged garlic extract also contains other antioxidants,albeit at a lower concentration, and it is likely that these make acontribution towards the anti-glycation effects observed in thisstudy. It might therefore, be preferable to use aged garlic extractin clinical studies as opposed to pure S-allyl cysteine. Indeed,clinical studies have shown that aged garlic extract protectsagainst atherosclerosis by preventing hypertension, reducingserum cholesterol and triglycerides and by inhibiting plateletaggregation and low density lipoprotein oxidation (Banerjee andMaulik, 2002; Rahman and Billington, 2000; Dillon et al.,2003). Furthermore, aged garlic extract enhances activity ofantioxidant enzymes such as superoxidase dismutase, catalaseand glutathione peroxidase, all of which protect against freeradicals (Wei and Lau, 1998).

No severe side effects have been noted in more than 40clinical trials using aged garlic extract. These studies, togetherwith a long history of human consumption, confirm the safety ofgarlic preparations, which are well tolerated, even in highdosages (Lau, 1987; Sumioyoshi et al., 1984). Both aged garlic

extract and S-allyl cysteine have numerous beneficial effects,some of which may be due to their anti-glycation properties.They deserve more attention as possible cost-effective, non-toxic candidates for use in delaying or preventing thecomplications of diabetes and ageing.

Acknowledgements

We would like to thank Universities UK for providingMuhammad Saeed Ahmad with an Overseas Research Students(ORS) award for his PhD studies. We are also grateful to theGerman Academic Exchange Service for providing him withthe DAAD Scholarship enabling him to work with Dr MonikaPischetsrieder at the Friedrich Alexander Universitat inGermany. We are grateful to Mick Hoult for his assistancewith the image analysis and with the preparation of thismanuscript.

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