Expression and enzyme activity of tyrosinase, TRP-1 and TRP-2 after UVB irradiation

ESDR I JSID I SID Abstracts

0595 ANALYSIS

OF THE

SECRETION

ROLE

OF SMALL

OF MELANOSOME

Wawmaka

GTP

IN PIGMENT

andMaramitsu

Kobe Umverslty

School of Medxme,

Melanosomes melanosomes

are producedand are rrensfered

of melanocytes.

accumulated

small GTP bmdingprotem

that has beenlmphcated

membranes

So. we inveslgated pigmenl methods.

Immunoelectron

the melanosomal locahzed

whether

R&A

ln conjunction

wth

melanosomal

membranes possibly

playmg

m

ofDermatology,

protan

that

~processes

that I&b protein

neuronal

cells.

plays

Rab3A

IS a

transport

that Rab3Awas

IS arsoclated

wrth

and confocal

analybis

resulls

pennuclear

Ii is known

detected

on

tolls.

melanosomes These

IN

Tatsu,~

inlntracellularvesicle

nucroscopy

mwoscopy

,n penphery

periphery,

revealed

m synap~c

showed

showed

on pennuclear wbcate area

a role m melanosomal

melanasomes

and

,n

mxnunofluorescense

that Rab3A 1s localized

membranes.Confocal~mmunofluorescense

melanosomes

A&l.

m melanocytes

We have reported m pigment

cells usmg nnmunoelectron

RABBA

from t,he tip ofdendntx

evidences

transport

ofnellrotransmlttcrs.

Bash1

Department

fully developed

vex&

the melanosoma1

CELLS

&ih&,

a key role of intracellular

and secretion

PROTEIN

Kobe, Japan.

mto keratmocyles

Recently.

BINDING

area,but

that

Rab:jA

dissooate

transport

on

that Rab3Ais ~5:apart once from

m pigment

from

binds

TO

them

,n

cells.

0598 M VITRO

MODEL FOR STUDYING EPIDERMAL CULTURES FOR TREATMENT OF VITILIGO. 3E Piam iati A Andreassi P Ta Andmsri.Dcpanment of Detmatoloey, ‘Department of Histology, University of Siena, Siena, Italy ITALY In the last few years SurgxaJ treatment of vitiligo has been found to be a belpftd tbempcutic approach in selected, stable cam of vl~ligo. In MLTDeprtment fin patients with localized, unresponsive vitiligo were treated with good rwdts by gratliog autologous epidermal cultures grown on perforated hyabronic acid (HA) membranes on previatrly abraded achromatic skin. In order to evaluate re.pigmentauon and re-&ixlization at various stages, we dcv&ped an in vitro excerimeotal model usinn deaidamiled human dermis IDED). Human cultwed eoidermis on p&orated HA nxmbran& was &ioned on the DED at c&h&ce, and cuhut’ed at tiie air-liquid intetfacz The results were evabuted by histology, immwobistochemLary and elenron microscopy aRer 3,6,9 and 13 days. Histological studies revealed that basal and sopmbasal ka&wyta arid melanccytes migrated tbmugb the holes of the membranes and disttiibuted physiologically on DW organizing in a multi&red epithelium The wface lsysr of the epidemda cukwed on the membranes did nca migrate but b&wed as a prowctive layer abaw the wc-epidemds. Transmission electmn micmsmpy showed lo-12 layers of kemtin~ts, with uhmstmw cbarxteritic~ similar to skin in viva. Basal keradnocytes, mntaining clusters of mekmosomes. adbad to the DED by hemidesmasomcs. Melanccytcs became multidendrhic, resem&tg normal mature melanwytes. fmmuno-staining with DOPA and s-100 coofirmed the &astmchual data and revealed the distribution of melmwytea among basal keratinocyts, on DED. ImmunochemicaJ study with Monoclonal Ab AE2 rev&d positive staining of the s.opmb& keratincxz+ layers, revealing a polarized different&n pattern. There results showed that the model is suitable for studying teepithelization and migration of melanocytes through the holes of the HA carrier onm the wound bed. It will make u pwsible to study the sffect of agents, such as W light, on the synthesis and transfer of pigment.

0596

0599

SUCCESSFUL TREATMENT OF EPIDERMAL HYDROGEN PEROXIDE (HtOz) STRBSS IN VITILIGO WlTH A TOPICAL PSEUDOCATALASE. m, D.J. Iman?. ‘Clinical and ExperimataI Dermatology, De&m& of’ Biomedical Sciences, University of Btadford, UK. ‘VitJlino Cbnic. Dwt of?&aillc-Facial Somew Uoiwa-sitv of Creit&ald. Ommanv. Vii is &w&red depigmmt&im d&&t affediogO.54% ofthe~orldpq&datic~ lb6 aetiology oftie dissaseis still Imknowo. Now&y3 there is sccumulatisg evidence for in-se4 HzO, pmdoaion in the eatire epidamis of a&ted individuals. This increased gmezuicm of HzOz C‘BIIbe attributed at leaat to threa major pathways: I. increased epidennal de now, synth&s/defeuive recyciii ofthe mfactot (6R) L-xythro 5,6,7,8 tamhydmbicpterin (6B&), 2. &eased epidennal MAO-A activity and 3. &teased epidermal nitric oxide s)athase activity. Patients WI& vi&go have low epidemml atalase levels in their involved and uninvolved epidermis althcugb the mRNA exptwica is normal. In ation a perttubed caloium hommstasis has been identiiied in bc& melaoocytcs and keratioocytes Bared on thea obsenaioos a pseu&xatalase., in wmbinatia~ with calciom, has proved socass~ in ths treatment of this disease. Utiliiing Fourier Transform Raman spectroscopy we &&ied I” v&o pidermal Hz& accumulati~l at 875 ati’ in patieuts skin, followed by its reductia~ a&r pseudocatalase application. In add&a 3 mm punch biopsies were obtained fmm 10 patimts involved and tiwlwd skm Wore, 6 and 12 mmths after treatment to assess the pressnca and number of foncticming m&oocyw using melaxomal markers (NKI B&b, HMB45), melar!jn staining @ntaoa Mass@ and tymskmse activity (dopa omdase) Photographic douunmtatim~ was utilised to demmmtrate vacuoIation iodutive of oxidauve stress in melangnes sod kemtmocyt~. The malts proved: 1. pseudcatalase reduces epidetmal HzO, directly within two minutes with a 15 fold farter tumovar canpared to natural catalase, 2. pseadcatalase prevmts vacuokatim in melanocyles and keratinoc,w and 3. pseudocatalase increases the number of fimctioning melaowytes in involved epidermis ofpatients with ntihgo.

CLINICAL AND PBYSIOLOCICAL FINDINGS IN TEE SKIN OF PATIENTS WJTH I’BE COMMON MELAS MUTATION M MITOCEONDRUL DNA S-L. Karvonen K-M. Haaoasaari. M. Kallioinw A. Oikminen. I.E. Hassinen and K. ullamaa ~eparunenu of Dermatology, Pathology and Nemology, University Hospiti of Zulu and Ihe Department of Medical Biocbadsy, University of Zulu, Oulu, Finland The MELAS syndrome (mitochontial encepb&myop&, lactic acid‘+ stroke-like episodes) is one of the mitwbondrial diwrdets. The most cantnon molecular ehology of this syndrome is a mutation at hp 3243 in the mitcchondrial genome (mtDNA). The clinical phmotw is variable and in awioo to the wti nervous system inv&.aem, MELAS wJe* may Prefent with neurosensw hearing loss, diabetes and cardmmyopatiy We wanted m smdy the pxsible involnment of tJx skin m this syndrome Cliructi evaluation of 28 ~titisnts with the bp 3243 mutation m mtDNA revealed vitiligo in 3 oftbe 28 patients (,I%), which is markedly more than in the general populauon (relative risk 28, 95% CI 9.6 to 83). bmnunobistcchemistly of affected skin samples showed absence of pigment, bw normal &triition of melanwytes in dermoepidcrmal junction. Also seborrheic eczema (29%, 95% Cl 13 to 49) and atopic ~nsotutmn (43% 9.5% Cl 25 to 63) were more freqwd in patients wtb MELAS mutation Using non-invasive sonogmphy and Iaser-Doppler flowmeoy we examined the thicknw sod blmd Bow of the skm. Collagen synthesis was assayed from a suction blister fluid Evaporimnry was used to assess the n-epitbelization rate of the suction blister wounds. Although the gewal appearance of the pauents with The swetc dudcal phenotype was slightly or conspicuously older than their age, their *in thickness, bkcd Ilow or the skin collagen gmthesis did tlot differ fmm the

0597

0600

EXPRESSION AND ENZYME ACTIVITY OF TYROSINASE, TRP-1 AND TRP-2 AFTER UVB IRRADIATION. Eri Nishioka. Yoko Funa.&_ I .“O’. Ashok Kumar Chakrabortv. K nd h Yutm Masamitsu Ichihashi. Department of Dermatology, Kobe Univarsity School of Medicine, Kobc, Japan. ‘Mishima Institute for Dermatological Research, Kobe, Japan. UVB activates melanin production by increasing tyrosinase activity. Tyrosinase-related proteins, TRP-1 and TRP-2, ate also involved in melanin formation, and play a role in protecting cells from death. To elucidate a role of tyrosinase, TRP-1 and TRP-2 in UVB irradiated melanocytes, the expression and activity of these proteins were studied iu relevance to melanin production, cell growth, and cell death. Among two lines of B16 mouse melanoma and six lines of human melanoma ( hMel ) , and wo normal human melanocytes ( hMC ), two B16, one hMel, and two hMC showed enhanced melanin conteot with increment

INCREASED

of tyrosinaae activity after 2.5-30 mJ /cm2 of UVB iradiation. Cell number and TRP-2 activity of these two 816 and one hMel lines decreased simultaneously. Further apoptotic change was observed 24h after UVB irradiation in these call Ilocs. TRP-2 transfection in one hMe1 rescued UVB-induced apoptotic changs. Taken together, TRP-2 might play a role in pro~ccting the pigment cell from UVB-induced apoptosis.

W”tI”lS ~3” prscnt study suggests that patiees with the bp 3243 mulation in NDNA have ioaeased prevalence of vitiligo However, the mclanwytes appeared normal in the ntiligo lesions suggesting a distu&anoe in synthesis or transport of melanin fmm the mdawxyies to the kcntinwytes. No fcatunS dpremahlre aging, such as marLed decrease m slun thickness, bled flow. collagen synthesis or mepilhelization rate, were demonstrated

EXPRESSION

OF AMINOPEPTIDASE

N (APN,

CD13)

AND

NEUTRAL ENDOPEPTIDASE (NEP, CDlO) IN PRIMARY MALIGNANT MELANOMA. T Boaenrieder. stolz. Department of Dermatology, University of Regensburg, Regensburg, Germany. Cell-surface peptidases are widely distributed in human tissues and function to hydrolyze peptides in the extracellular space, including peptide hormones and growth factors. They also have been implicated in the degradation and invasion of the extracellular matrix (ECM) by metastatic tumor cells. We used peptidase-specific monoclonal antibodies (mAbs) and immunohistochemical techniques to determine the expression pattern of aminopeptidase A (APA; mAb S4). aminopeptidase N (APN, CD13; mAb F23) and neutral endopeptidase (NEP, CD10; mAb J5) in cryostat sections from 5 benign nevi as well as 11 primaly and 1 metastatic malignant melanoma (MM). APA immunoreactivity could not be detected in any of the investigated benign or malignant melanocytic lesions. However, benign nsvi demonstrated APN and NEP expression in 3/5 and 2/5 specimen, respectively. In contrast, APN was expressed in r50% of tumor cells in 12/12 MM specimens. NEP was expressed by >50% of tumor cells in 1 l/12 of primary MM. These data show that expression of APN and NEP is increased in MM, Increased expression of these cell-surface peptidases presumably would either activate precursor forms of biologically active peptides to interact with MM cells or degrade inhibitory peptides. Alternatively, they could be directly involved in ECM degradation and thus potentially affect MM growth and metastasis.