LINK 22.doc

Link 22-BCM stories  

 

To the chemistry student who like Nanoscience

Useful for thesis preparation on melanin

Under construction

 

www.tightrope.it/nicolaus/index.htm

www.organicsemiconductors.com         

www.brunonic.org 

 

 

 

          

Granules of Rana pipiens skin (taken from G.Moellmann, J.McGuire, A.B.Lerner, The Yale Journal of Biology and Medicine, 46, 337-360, (1973). On the right Tyrosine-melanin granules (taken from M.R.Okun, R.P.Patel, B.Donnellan, L.M.Edelstein, Proceedings of the 8th International Pigment Cell Conference, Sydney, March, 12-17 (1972). The granule is a melanosome in which any chemical or enzymatic activity is spent.

----------------------------------------------------------------------

 

 

.

The black material is formed of  particles. The particles are constructed by oligomers formed from precursors which are generally  O. diphenols. The oligomers of DHI-melanin, DHICA-melanin and sepiomelanin were found of low molecular weight (43), (44), (45). Optically active oligomers were recently discovered (103).Cis/trans isomerism is showed by acetylene-black and pyrrole-black. Isomers have different conductivity

---------------------------------------------------------------

The centesimal values for a polyindolequinone structure  ( C8H3O2N ) formed following the  Raper-Mason  scheme  are  :

C%   66.2   H%   2.1   N%   9.6

-------------------------------------------------------------------

The DHI-melanin analysis ( R.J.S.Beer, T.Broadhurst,A.Robertson, '' The chemistry of melanins-Part V-The autooxidation of 5,6-Dihydroxyindoles '' Soc. 1947 ( 1954 ) gave :

C%   56.6   H%   3.1   N%   8.2

which approximates to the formula  ( C8 H5 O3 N ) which may be explained with addition of water to a carbonil group, presence of carboxylic groups,storage of water or oxygen.

-------------------------------------------------------------------

 The values of BCM ( natural melanins ) were  different as for the ink of cephalopds

C% 54.3  H%  2.9  N%  8.8

calculeted for C8 H7 NO3     C% 58,2 H% 4,2 N% 8,5

                        C9 H7 NO5     C% 51,7 H% 3,3 N% 6,7

(J.P.Ortonne, C.Voulot, C.Khatchadourian, A.Palumbo, G.Prota '' A reexamination of melanogenesis in the ink gland of cephalopods '' in Pigment Cell 1981 page 49-57,Ed.M.Seiji, University of Tokyo Press, 1981. )

 but may approximate the centesimal value for C8H3O2N ( depending of physical methods of purification adopted ( 62a), (62b).

-------------------------------------------------------------------

 Melanin  may be degraded in mild conditions by H2O2  ( in vitro and in vivo ), or  photooxidatively   by ultra violet light. Typical the fragmentation which occurs in the MALDI experiments. It is interesting that the colour changes  (black, reddish, gold yellow ) during the H2O2 oxidation corresponds to those of some inorganic sulphides (8 ). The colour depends on the gap amplitude of the semiconductor model ( 8 ), ( 27d ).  ( Fig. 1 ) (Link 21).

 Natural and also synthetic melanins particle are paramagnetic   because they contain unpaired electrons within their molecules ( see acetylene-black) and are amorphous semiconductors : ( www.organicsemiconductors.com ;  www.tightrope.it/nicolaus/index.htm  ), ( 27 a-d), (27h), (27l ) (27m ).

The EPR spectra of natural melanins (BCM) and black  synthetic materials   (BSM) are quite similar .The EPR spectra  cannot distinguish between these pigments which we know are in some ways  chemically different ( catechol-melanin, dopa-melanin, cyclodopa-melanin, DHI-melanin), that is the EPR spectra are of no relevance for  melanin  structure of precursors. 

 The EPR results raise significant questions : first the EPR signals ( electrical conductivity ) which one invariably observes  in BCM and BSM, are not seen in any other biochemical compounds, or in tissues or organisms not containing melanin.

Thus melanins ( BCM ) seem to be unique among the costituents of living organisms  in that they exhibit  intrinsic semiconductivity and have characteristic band gaps 1,2-1,7 eV (black of the semiconductor model ) , gas-storage properties,  particle structure and binding property ( binding effect ) for ions, organic substances, reactants, solvents (3), (4 pag. 78-80 ), (5), (12), (20), (30), (78-82)..

The radical-polarone (cationic) system seems to be present in all the BCM and BSM until now examined.The models explain a number of properties like optical, magnetic, electrical, chemical, assembling of oligomers. (27 b-d ). The description of neutral and charged defects in conjugate polymers is based on Huckel calculations which have quantitative limitations. More sophisticated treatment of neutral and charged defects in polymers differ from Huckel description in quantitative but not in qualitative conclusions.Whereas Huckel treatment  of a radical defect on polyacetylene yields a distortion of the lattice extending about seven carbon atoms on either side from the defect, detailed calculations yield an entity localised on three carbon atoms.

Secondly the EPR absorption and other properties, which melanin alone appears to have in living organisms, are found abundantly among products of non-living  systems ( BSM ) including probably black interstellar matter (27f), (27g). 

 (Link21).

  Interaction of radical and cationic centers or chirality of the oligomers may intervene in the assemblage process.

BCM particle is made up of small oligomer units ( 102),  ( 43-47 ), (11), ( 10 ),  and  the structural morphology reflects the aggregation of  oligomeric molecules.

 Absorption of gas, liquids, organic substances can affect elemental analysis, IR, NMR, MALDI spectra. Absorbed diazomethane may affect the methoxy value and binding oxygen can mask hydroxylic and carboxylic groups..

The electrical conductivity of a black substance may be notably increased by doping ( as in  acetylene- black and pyrrole black ) (28-30 ).  Melanins produced synthetically and isolated from biological systems act as amorphous semiconductors with threshold switching. Switching is reversible at potential gradients two to three orders of magnitude lower than reported  for inorganic thin films  and comparable to gradients existing in some biological sources.  Melanins give off a flash of light when they switch ( electroluminiscence ) ( organic semiconductors.com   ) Often the study of melanins (BCM and BSM) is carried out on artefacts. Artefacts of melanins, little known, from a chemical point of view, formed in the course of the synthesis or extraction from the biological sources, even in the blandest of conditions: processes of oxidation, decarboxylation, condensation, or substitution of the natural cation may all take place during purification 

It should be remembered that all the o.diphenols may produce melanin and H2O2 in the presence of  oxygen ( cellular or atmospheric ) . Black material (melanin) is formed by oxidation in atmospheric O2 or by H2O2 especially at elevated pH in not reproducible yields. The material always has one oxygen more than the number present in the precursor (catechol, 5,6-dihydroxyindoles, adrenaline, (Link 15), DOPA , dopammine, methyl-catecholes and methyl-5,6-dihydroxyindolesetc. ).( Fig.5-8).

Starting from tyrosine or DOPA by enzymatic or chemical oxidation a red solution (the assembling moment of oligomers) is obtained containing many species of quinones and phenols.The red solution is called dopachrome and any substances which decolorize the solution is called enzyme.

Attempts of extraction and purification of melanins by preventative mixing of the various organelles of the melanocyte have been made ( 31 ). The extraction of melanin from the various biological sources is, effectively, the extraction of granules and of active melanosomes and premelanosomes. The melanin which is obtained by the various extractive processes used to date is a set of particles partly formed during the course of extraction. The melanin which is obtained is, therefore, an artefact. The subsequent purification process  produces:

-------------------------------------------------------------- 

a. decarboxylation on heating

b. oxidation of the benzenoid part ( when the precursor is 5,6-dihydroxyindole, DHI ) by the H2O2 present in atmospheric oxygen

c. carbazole condensation processes with the use of hot HCl (  formation of pyrroletetracarboxylic acid in the oxidative degradation ).

.d.  binding  for reactants and solvents.

------------------------------------------------------------- 

As consequence of the melanin beaviour analytical results of ( 36 ) (85) obtained until now are doubtful . The study of pure granules fractions may be useful.

Wishing to summarise we may claim that to date nobody has managed to isolate a particle equal to that contained in a granule of melanocyte. It follows that all the experimental data and chemo-physical parameters are uncertain and need to be revised with particular  attention..

Effectively the black particles built with oligomers of non-high molecular weight ( 102 ), (11) explode (they break like a vase dropped from a height) under LASER action. The explosion is similar to that  of the graphite, which as is known, brought about the discovery of the fullerene C60 . It is possible that a similar reaction takes place because of the effect of an undocumented radiation or the collision of black interstellar particles.( Link 7. Speculation on the chemistry of interstellar black matter. or Link 8. The dark secret of life ).

Considering the biogenesis of the cellular pigment, it is believed that the melanins are polymers of DHI and/or DHICA (initially believed to be artefacts by Raper). The reactions which lead to 5,6-dihydroxyindole (DHI) and to 5,6-dihydroxyindole-2-carboxylic (DHICA) would thus be under the rigid control of several enzymes and other factors. As a consequence there should be an enzymatic system which regulates the construction of the black particle. It should be noted that among other things the sepia may also yield a melanin from a precursor other than the tyrosine, which certainly appears not to favour the notion of rigid enzymatic control of melanogenesis (37) (81-84).

Enzymatic systems, mainly belonging to pathological melanogenesis, which transform the dopachrome into DHI and DHICA have often been reported in the literature . Some studies (32-33), have found that there is a protein other than tyrosinase in the melanocytes which catalyses the conversion of the L-dopachrome at pH 6.8  into DHICA. Several names have been given to the enzymes and related substances like : a. DCF Dopachrome conversion factor b. ICF  indole conversion factor c. DOR  dopachrome oxido-reductase d. TYR  tyrosinase e. DI  dopachrome isomerase f. DCT,DT    dopachrome tautomerase (74), (49), (50), (51), (33),(49bis) g. TRP1   tyrosinase-related-protein 1 h. MIF  migration inhibitory factor. i. IQCA  5,6-indole-quinone-2-carboxylic acid j. MART 1   melanoma antigen recognized by T lymphocytes

DHICA produced either enzymatically with DCT or through chemical reaction polymerize to form brown melanins (BCM) soluble in alkaline solutions.Under the same conditions DHI, Cyclodopa,  DOPA form black insoluble material. The enzymatic activity was always based on the decoloration of the red solution, a decoloration which may be obtained with a plenty of inorganic and organic compounds.

The enzyme seems to be present not only in the melanocytes of melanoma but also in the melanocyte of sepia ink (34). A different enzyme which catalyses the conversion of the dopachrome into DHI seems to be present in insects (4). Analytic studies which demonstrate that in dopa-melanin the DHICA unit represents only 10% while in melanoma-melanin (35), only 20%  (36). An attempt to reconcile the action of enzymes with the action of metals  has  been presented (4).

 To explain the chemical data, in contrast with the melanogenesis scheme, we suggested that the eumelanins originate from o.diphenol cyclodopa whithout transformation into aromatic systems,  and that the fundamental unit  has hydrated quinone unit. As consequence  polymers and oligomers have one oxygen more than the precursor  .  The units which constitutes the black particle may be described as a radical-polarone system well represented by acetylene black  and having a typical EPR signal. The form of the black particle,  rather than the molecular structure, explains properties like the affinity for ions, organic molecules and gases, the ability to reduce molecular oxygen, to superoxide ions and hydrogen peroxide . The concept of the particle changes the way of studying the melanins (BCM and BSM) and takes us instinctively to investigate the monomers and oligomers which contribute to form  the black particles. The form and size of the particle may be a distinctive element of the property of different melanins.

Analytical data may change by binding effect for O2 , reactants like diazomethane and dimethylsulphate    In agreement with chemical analysis and in disagreement with literature (101) BCM is formed from DOPA and Cyclodopa (Link 20) and not from DHI or DHICA.

Remember that  :

 Melanin  is not a  polymer.

 Melanin is a particle  formed by radical-polarones of polyene systems the most simple one being acetylene-black.

Melanin is not a biological garbage as commonly believed.

Melanin is one of the most  important material involved in Birth  and Evolution of living matter.

Peculiar properties known until now  are:

  1. Sound and electrical conductivity    ( BCM and BSM )
  2. Storage of gas, water, organic and inorganic liquid and solid products .
  3. Electrical treshold switching

d.   Control of  shape and function of adherent cells………

 

Testo italiano. ( Per le figure riferirsi al LINK 21 ).

Le melanine hanno sistemi radical-polaronici responsabili del segnale EPR, della conducibilità e del colore del materiale. Una tale situazione elettronica è stata studiata da ( 63 ) ed applicata nel caso del nero di acetilene.Il sistema del nero di acetilene è estensibile a tutti i neri finora esaminati  (27b ).La melanina ( BCM e BSM ) è diffusa sulla terra e negli spazi interstellari ( 27f ) , ( 27g).  Oltre che per le sue proprietà assemblatrici tipiche delle BCM la forma idrata del indolchinone è atossica per la cellula e possiede molte delle proprietà di superfici del PLGA-collageno, le sue proprietà elettriche (effetto Proctor-McGinness) rappresentano un elemento di comunicazione fra tessuto e tessuto e fra tessuto e SNC. .Per quanto concerne i valori dei dati analitici questi possono essere modificati per il binding effect della particella verso acqua,  ioni, molecole, gas, reagenti come ad esempio il diazometano e il solfato dimetilico ed infine i solventi.

Microanalisi della BCM basata sui prodotti di degradazione ossidativa è stata descritta ( 67 ) , L' ossidazione permanganica dà l'acido pirrolico  ( 64 ) mentre l' idrolisi con acido iodidrico della feomelanina  produce amminoidrossifenilalanina  ( 66 ). (Link 14) Un altro prodotto che si forma talvolta nella ossidazione del pigmento con permanganato è l'acido 2,3,4,5-pirroltetracarbonico    .Secondo  ( 67 ) questo prodotto  è un artefatto.

Vi sarebbero secondo noi  due spiegazioni :

a).L'acido   viene perduto nel corso della estrazione per la sua proprietà di formare un sale monopotassico insolubile in tutti i solventi compresa l'acqua (65).

b).Una struttura carbazolica ( 68 ) può formarsi per azione del acido cloridrico su i 5,6-diosiindoli e quindi originarsi anche per  azione dello acido sul pigmento che a sua volta darebbe l'acido tetracarbossilico II per ossidazione .

La sintesi della BCM procede attraverso stadi diversi ( 70,71 ) rappresentati  da :

a)         vescicole intermedie ( 0.5 micron di diametro ).

b)         premelanosomi         ( 0.7 x 0.3 ).

c)         melanosomi              ( 0.7 x 0.3 ).

d)         granuli di melanina   ( 0.7 x 0.3 )

attraverso i quali i polipeptidi sono sintetizzati,le molecole enzimatiche sono ordinate.Vi sono granuli che conservano attività tirosinasica e nei quali è presente acqua ossigenata e granuli in cui nessuna attività è riscontrabile.Questi contengono la BCM. Nel corso dei processi estrattivi un pigmento di composizione e struttura artificiale rispetto alla BCM si forma. Perdipiù il granulo subisce modificazioni chimico-fisiche nei processi di purificazione e perfino nei processi di invecchiamento dei campioni così come è stato osservato in certi lavori MALDI sulla sepiomelanina (72 ) Il granulo (particella), possiede notevoli capacità di legare ioni, molecole, gas, reattivi,solventi. Da notare che nel corso delle pesate dei campioni si è osservato spesso una perdita di peso.Per tutti questi motivi i metodi analitici sono poco affidabili e in particolare quello proposto da ( 36 ) (85) . Tirosina e DOPA danno per ossidazione  enzimatica o chimica una soluzione rossa contenente diverse specie di chinoni e fenoli. La soluzione rossa può essere decolorata da sostanze organiche ed inorganiche le più diverse. La soluzione rossa (il momento dell'assemblaggio molecolare) non può perciò essere considerata come substrato di un presunto enzima. Di tutto ciò non si è mai tenuto conto negli esperimenti di laboratorio.

I precursori della BCM eumelanine  potrebbero essere oltre che DHI e DHICA anche  la DOPA e la ciclodopa  In questo caso le melanine sarebbero caratterizzate  da un  gruppo --CH2--  . Il dosaggio e l'analisi del metilene o gruppo  --CH2---, possibile con vari metodi fisici e chimici , rappresenta perciò un punto di passaggio e riferimento  per chi si accinge a studiare le melanine (BCM ) ed a classificarle.Il problema del riconoscimento e dell’attribuzione dei segnali NMR da 13C nella zona alifatica ha ricevuto un’attenzione sempre piuttosto scarsa e richiederebbe uno studio specifico per risolvere questioni lasciate aperte. Ad esempio, per quanto concerne i segnali attribuibili a gruppi CH2 , sempre broad,  tutti i lavori esaminati rilevano che la loro intensità è maggiore nelle melanine naturali (prevalentemente Sepia) rispetto alle sintetiche, giungendo generalmente alla conclusione che la presenza di  amino-acidi o proteine  sia la responsabile delle risposte. Poca attenzione viene data al fatto che il segnale è presente anche nelle melanine prodotte per auto-ossidazione ( dopa dopammina ).    Nel caso della  dopammina-melanina  la sua natura alifatica è ampiamente dimostrata     ( 69 ),   ( 69c), ( 69g ). In realtà, si rileva nella zona alifatica dei forti segnali ( melanina sintetica,  melanina da melanoma e melanina della Sepia ) ( 38 ). che vengono  ritenuti responsabili i  monomeri o i precursori non ciclizzati che non hanno reagito e sono stati intrappolati nel '' polimero''. Sia in questo lavoro, sia nei successivi, la regione tra 15 e 75 ppm contiene più risonanze mal risolte che non si prestano ad un’interpretazione inequivocabile, come ad esempio i picchi a d = 54, 36 e 31 degli spettri  (39-41) I risultati sono in accordo col modello di composizione statistica proposto da Swan.( 69  ) ed Hempel ( 77 ) in numerosi lavori con precursori segnati. .

Altri tentativi di interpretazione degli spettri NMR sono stati fatti comparando melanine e i loro cosiddetti "free acids" ( tutte le melanine naturali si trovano sotto forma di sali.Ad esempio la

sepiomelanina è un sale di calcio e magnesio. E' perciò meglio riservare il termine ''free acid'' alle melanine spostate dai loro sali  piuttosto che a quelle ottenute con processi ossidativi.   Di notevole interesse è  il lavoro  (42) nel quale l’intensità dei segnali dei carboni alifatici è valutata come il 20 % del segnale totale. Il teorico dei carboni alifatici del dopacromo è circa il 17%

Naturalmente lo studio è valido se  fatto su materiale ottenuto razionalmente e riproducibile dovendosi escludere la presenza di proteine ,di amminoacidi e precursori coinvolti nella sintesi della particella nera. Una strategia vincente potrebbe essere quella di separare i granuli di melanina più densi  evitando la formazione di artefatti .

Il riconoscimento del gruppo --CH2-- e il suo dosaggio ci  può dare utili informazioni sulla genesi da ciclodopa o da DOPA delle melanine e sulla loro natura amminoacidica..

Il gruppo metilenico è presente sia nelle melanine sintetiche ( dopa-melanina ) BSM che in melanine naturali ( sepiomelanina ) BCM. Perciò  dobbiamo ritenere che le eumelanine non sono costruite con il 5,6-diossindolo (DHI) o il 5,6-diossindolo-2-carbossilico (DHICA). Questi composti e i loro prodotti di ossidazione (altamente citotossici) possono essere considerati degli artefatti. E' come dire che la ciclodopa produce il nero senza convertirsi  in DHI o DHICA.

Analoghe sono le formule del tetramero del catecol-chinone e del DHI chinone nelle forme idrate. Si noti come i monomeri, in accordo con le analisi centesimali, hanno un ossigeno in più dei corrispondenti precursori ( adrenalina, catecolo, 5,6-diossiindolo etc. ) Fig. 7 e 9.

Come si vede dalle figure le unità contengono un ossigeno in più rispetto al monomero.Noi abbiamo spiegato questi dati con l'idratazione di un gruppo chinonico. ( 27b ), ( 27h ),  ( 27m ).ma un binding effect dello ossigeno non può essere escluso. La ciclodopa ( 73 ) è una sostanza molto sensibile all'ossigeno  che può dare un nero senza convertirsi in DHI  o DHICA  in accordo con quanto osservato fin dai tempi di Raper (2). La presenza di unità dopacromiche  o ciclodopiche è stata spesso segnalata    (3 ), (38), (40-42),  (15), ( 27d ), ( 27h ), ( 27m ). Dunque le melanine sembrano avere sempre un ossigeno in più rispetto al precursore (7) (vedi Fig. 7 e 9 ). E' da notare che la presenza di triossiindoli,  rivelata con la spettrometria MALDI,  è stata riportata in un lavoro sulla melanogenesi ( 11 )  e che una melanogenesi basata su precursori triossigenata fu proposta fin dal 1989 da un gruppo di americani ( 7 ).  Il riconoscimento della natura di particella della BCM ( black cellular matter, melanina, eumelanina, neuromelanina, acidi umici etc ) la quale non ha ovviamente peso molecolare ma viene convenientemente descritta  con la forma , grandezza e struttura interna.Parametri che possono caratterizzare il comportamento chimico e proprietà chimico-fisico quale coducibilità, assorbimento dei gas, sequestro di ioni e molecole organiche,influire sulle funzioni biologiche come la capacità di assemblaggio molecolare e macromolecolare. ( funzioni della era post-enzimatica ). I nuovi concetti richiederanno certo del tempo per risolvere l'attuale grosso pasticcio in cui si trova la ricerca ma essi risolvono molti misteri e permettono   la soluzione del problema. Direttamente ed indirettamente   si è potuto ancora stabilire che la costruzione della particella, in sostituzione del vecchio polimero richiede solo oligomeri ottenuti da  o.difenoli anche molto diversi fra di loro ( Dopa, ciclodopa, catecolo, 1,8-diossinaftalina, DHI, DHICA, diossichinoline ed isochinoline etc. ) : Molti dati indicano che la particella nera è costruita con oligomeri di peso molecolare non elevato (102), (4), (11), (10),  (41-44).   Oligomeri di precursori diversi, cioè miscele di precursori costruiscono la particella nera  ( BCM, BSM ) e possono influire su grandezza forma e proprietà fisiche .Quali  siano i meccanismi di assemblaggio   non lo sappiamo ancora.

Il LASER  (condizioni MALDI e MALDI-TOF ) frantuma la particella nera mostrando i suoi oligomeri e prodotti della esplosione del sistema ( su ciò si basa la depigmentazione e depilazione dei peli neri usata in dermatologia, nonchè la tecnica recente di pulitura delle opere d'arte ). Picchi relativi al materiale nero non sono osservabili al MALDI e MALDI-TOF. Che la particella nera sia formata da oligomeri  a basso peso molecolare si può anche dedurre in modo indiretto dallo esame di melanine sulle quali siano stati assorbiti i prodotti di ossidazione degli oligomeri formatesi nel corso della sintesi ( 43-47  ).La prima comunicazione che fà esplicito riferimento ad una particella risale al 1992 ( 9 ) . La prima osservazione che la melanina è formata da gruppi omogenei di oligomeri risale al 1993 ( 102 ).La prima comunicazione riguardanti gli oligomeri a basso peso molecolari quali mattoni della particella nera, cancellando la credenza del polimero, è del  1995 ( 46-47 ). Dunque la nanofisica, la nanochimica, la nanobiologia della BCM sono  materie giovani.

Interessante per la formazione delle particelle nere la recente scoperta di oligomeri otticamente attivi (103).

 

Una melanogenesi enzimatica per formare   materiali amorfi sembra poco credibile.Inoltre la tirosinasi agisce anche su altri fenoli che abbiano una posizione orto libera. producendo sia in vivo che in vitro materiale nero. (37), (83), (84) Inoltre gli o. difenoli si convertono spontaneamente e rapidamente in materiali neri o bruni per la formazione dei quali non sembrerebbe necessario accelerare la velocità di reazione con l’intervento degli enzimi. Noi guardiamo piuttosto alla esistenza di sistemi assemblatori degli oligomeri per la costruzione della particella e allo straordinario sistema elettronico presente negli oligomeri. 

 

In breve   :

Tutta la materia nera è caratterizzata dallo stesso sistema elettronico. radical-polaronico che è anche responsabile di molte proprietà fisiche.La melanina ha un ruolo fondamentale nella  evoluzione della materia vivente

a.         La materia nera della cellula ( BCM ) è derivata dalla DOPA e/o dalla ciclodopa o dai loro prodotti di ossidazione dopachinone e dopacromo prodotti non isolabili allo stato cristallino per la loro instabilità (produzione di materiale nero ) . Ciò è sperimentalmente dimostrato dalla presenza nella melanina di unità caratterizzate dal gruppo metilenico ( parte alifatica degli spettri 13C NMR ) presente solo nella dopa/ciclodopa-melanina e non nella DHI/DHICA-melanina.  (37 ), ( 38 ), ( (62 ), ( 69 ), ( 69 a ), ( 77 ).

b.  E' stato sperimentalmente provato che il melanoma è fatto da una melanina bruna e da una melanina nera brillante ( 61 ) che sembrano corrispondere alla  DHICA-melanina e alla DHI-melanina  ( 61 ), naturalmente in forma chinonica idrata .  Ciò suggerirebbe l' esistenza di una melanogenesi patologica ed una fisiologica.

c.   Solo l'ossidrilazione della tirosina è una reazione enzimatica .

d.   Tutte le analisi, eseguite correttamente, mostrano che la melanina ( BCM ed BSM ) ha un ossigeno in più rispetto al precursore ossigenato. Ciò può essere spiegato o con l'addizione di H2O al chinone o per binding effect dello O2. 

e.   La chimica indica per gli oligomeri una struttura radical-polaronica (  vedi  nero di acetilene ) .

f.    Gli oligomeri ( 11 ), sono assemblati in particelle nere che si possono, anche in condizioni blande,  frammentare  ( vedi la grafite )  con il LASER (MALDI) o per via chimica con acqua ossigenata.

Con la formazione di un nero da ciclodopa diversi problemi chimici e biologici inerenti alla BCM potrebbero essere chiariti. ( 76 ) .Oligomeri radical polaronici a basso peso molecolare si assemblano per formare la particella. E' probabile che forma, grandezza, struttura possano influenzare la conducibilità elettrica e sonora, il trasporto di gas, ioni, molecole organiche sia della materia nera cellulare (BCM) che della materia nera sintetica (BSM ). Non è sicuro che la melanogenesi proceda attraverso fasi controllate dagli enzimi essendo più probabile l'azione esercitata da metalli quali Fe, Cu, Zn, Ni.  La presunta attività enzimatica è dosata sulla decolorazione , opinabile, della soluzione rossa.  Lo studio chimico è carente di analisi C, H, N, S, Cu, Fe, Zn, Ni, e degli spettri di massa. Per quanto riguarda la funzione della BCM ( melanine naturali ) ,una questione assai dibattuta, non si è mai tenuto conto di quanto la chimica e la fisica venivano man mano  scoprendo. La conducibilità elettrica e sonora, la proprietà, comune a tutta la materia nera, di legare ioni, molecole organiche,  gas, la proprietà di esplodere della melanina e del melanosoma  (59 ) per azione  del LASER , la proprietà di assemblaggio molecolare ( principio della tensitegrità), la straordinaria  sensibilità alla acqua ossigenata. Infine la conducibilità e la superconducibilita a vari valori del potenziale (effetto Proctor-Mc Ginness ) www.organicsemiconductors.com.  

Gli studi chimici sulle melanine patologiche sono scarsi. Un vecchio lavoro fatto a Napoli (61) riferisce sulla estrazione da un melanoma di due melanine una nera brillante e l'altra matta, differenti per solubilità in alcali, che potrebbero essere una miscela di DHI-melanina e DHICA-melanina. E' possibile che DHI e DHICA siano coinvolti nella melanogenesi patologica e non in quella fisiologica.

                                             BIBLIOGRAPHY

Papers which do not report pigment chemical  ( C,H,N,S, Cu, Ni, Fe, Zn, for sepiomelanin Ca and Mg also ) , NMR, MALDI, analyses should not be accepted for publication

1.H.S.Mason '' Structure of Melanins'' in Pigment Cell Biology pag.563-581, Ed.M.Gordon, AP , New York 1959.

2.R.H.Thomson '' Melanins '' in Comparative Biochemistry pag. 727-750, Ed. Florkin-Mason, AP New York and London 1962

3.R.A.Nicolaus '' Melanins '' pag..1-305, Chemistry of Natural Products, Series Edited by Edgar Lederer, Hermann Paris 1968.

4.G. Prota "Melanins and melanogenesis"  pag. 1-290 , AP, San Diego 1992

5. G. Prota "The Chemistry of melanins and melanogenesis" in progress in the Chemistry of Organic Natural Products Ed. W. Herr, G.W. Kirby, R.E. Moore, W. Steglich, C.H. Tamm, pag. 113, Springer Verlag Wien New York 1995 ; Pigment Cell Research 13, 283, 2000.

6.P.A.Riley  '' Melanin '', Int.J.Biochem.Cell Biol., 29, 1235-1240, 1997.

7. R.A.Nicolaus, A.Bolognese, B.Nicolaus '' The Pigment Cell and its Biogenesis '' Accademia   Pontaniana 2002.

8. Le Scienze quaderno n° 21, 1985 , pag. 69.

9. L.Zeise, Brown L.Murr, M.R.Chedekel ''Melanin Standard Method : Particelle Description '', Pigment Cell Research, 5, 132-142, 1992. ; Pigment Cell Research Suppl. 2, 48-53, ( 1992 ).

10.C.M.R. Clancy, J.B.Nofsinger, R.K.Hanks, J.D.Simon,’’  Hierarchical self-assembling of Eumelanin ‘’ J.Phys.Chem. 104, 7871-7873, 2000 ;  C.M.Clancy, J.D.Simon, '' Ultrastructural organization of eumelanin from Sepia officinalis measured  by atomic force microscopy '' Biochemistry, 40, 133-153, (2001) ; Y.Liu, J.D.Simon ‘’ The effect of preparations procedures on the morphology of melanin from ink sac of Sepia officinalis ‘’ Pigment Cell Research 16, 72-80, 2003.

11. C.Kroesche, M.G.Peter '' Detection of melanochromes by MALDI-TOF mass spectrometry '' Tetrahedron, 52, 3947-3952, ( 1996 ).

12. Cope, F.W.: Eye melanin free radical kinetics and mechanism in relation to the Roginsky-Zeldovich (or Elovich) equation and the adsorption of oxygen by semiconductors. J.Chem.Phys. 40 (1964) 2653-2656

13. McGinness, J.: Mobility gaps: a mechanism for band gaps in melanins. Science 177 (1972) 896-897

14. McGinness, J., Proctor, P.: The importance of the fact that melanin is black. J.theor.Biol.39 (1973) 677-678

15. Crippa, P.R., Cristofoletti, V., Romeo, N.: A band model for melanin deduced from optical absorption and photoconductivity experiments. Biochim.Biophys.Acta 538 (1978) 164-170

16. Zeise, L., Murr, B.L., Chedekel, M.R.: Melanin standard method: particle description. Pigment Cell Res. 5 (1992) 132-142

17. Sarna, T.: Properties and function of ocular melanin. A photobiophysical view. J.Photochem.Photobiol.B: Biol. 12 (1992) 215-258

18. Chedekel, M.R.: Photophysics and photochemistry of melanin. In: Melanin: its role in human photoprotection (Zeise, Chedekel and Fitzpatrick, eds.) pp. 11-21 (1995)

19. Menter, J.M., Willis, I.: Electron transfer and photoprotective properties of melanins in solution. Pigment Cell Res. 10 (1997) 214-217

20. Crippa, P.R.: Oxygen adsorption and photoreduction on fractal melanin particles. Coll.Surf.B: Biointerf. 20 (2001) 315-319

21. Thathachari, Y.T., Blois, M.S.: Physical studies on melanins. II. X-ray diffraction. Biophys.J. 9 (1969) 77-89

22. Chio, S.S.: X-ray diffraction and ESR studies on amorphous melanin. Ph.D. Thesis, Univ. of Houston (1977)

23. Bridelli, M.G., Crippa, P.R., Ugozzoli, F.: X-ray diffraction studies on melanins in lyophylized melanosomes. Pigment Cell Res. 3 (1990) 187-191

24. Cheng, J., Moss, S.C., Eisner, M., Zschack, P.: X-ray characterization of melanins-I. Pigment Cell Res. 7 (1994a) 255-262

25. Cheng, J., Moss, S.C., Eisner, M.: X-ray characterization of melanins-II. Pigment Cell Res. 7 (1994b) 263-273.

26. Zajac, G.W., Gallas, J.M., Cheng, J., Eisner, M., Moss, S.C., Alvarado-Swaisgood, A.E.: The fundamental unit of synthetic melanin: a verification by tunnelling microscopy of x-ray scattering results. Biochim.Biophys.Acta 1199 (1994) 271-278

27

a.         R.A. Nicolaus, '' Divagazioni sulla struttura a banda del colore in natura : il nero '', Rendiconto della Accademia delle Scienze Fisiche e Matematiche, Vol. LXIV, 145-216,  (1998 ).   

b.         R.A.Nicolaus, A.Bolognese, B.Nicolaus '' The Pigment Cell and its Biogenesis '' Accademia Pontaniana 2002.

c.         B.J.R.Nicolaus, R.A. Nicolaus '' Speculating of the Band Colours in Nature '' Atti Accademia Pontaniana Vol.XLV, 365-385, ( 1997 ).

d.         R.A.Nicolaus, G.Parisi '' The Nature of Animal Blacks '' Atti Accademia Pontaniana, Vol.XLIX, 197-233, ( 2001 ).

e.         G.Nicolaus, R.A.Nicolaus ''Melanins, Cosmoida,Fullerenes '' Rend.Acc.Sci.Fis.Mat., Vol.LXVI, 131-158, ( 1999 ).

f.          B.J.R.Nicolaus, R.A.Nicolaus, M.Olivieri '' Riflessioni sulla materia nera interstellare '' Rend.Sci.Fis.Mat., Vol. LXVI, 113-129, ( 1999 ).

g.         B.J.R.Nicolaus, R.A.Nicolaus '' Lo scrigno oscuro della vita '' Atti Accademia Pontaniana Vol. XLVIII, 155-180, ( 2000 ).

h.         Comunicazioni Brevi, Atti Accademia Pontaniana, Vol. V, 391-404, ( 2002 ).

i.          R.A.Nicolaus,G.Scherillo, '' La Melanina.Un riesame su struttura,proprietà e sintesi '', Atti Accademia Pontaniana, Vol.XLIV, 265-287, ( 1995 ).

l.                      R.A.Nicolaus '' Coloured organic semiconductors '', Rend.Acc.Sci.Fis.Mat., Vol.LXIV, 325-352, ( 1997 ).

m. Comunicazioni Brevi, Atti Accademia Pontaniana Vol.XLIX, 301-312 ( 2001 ).

n.         R.A.Nicolaus, G.Misuraca '' Colore 90 '' Atti Accademia Pontaniana, Vol.XL, 83-107, ( 1991 ).

o.         R.A.Nicolaus '' Melanine '' Quaderni della Accademia Pontaniana n° 4, 1-53, ( 1984 ).

p.         R.A.Nicolaus,E.Novellino, G.Prota ''Origine e significato del colore negli animali '', Rend.Acc.Sci.Fis.Mat., Vol. XLII, 1-82, ( 1976 ).

28.  G.A.Pagani, G.Gardini, ''I metalli organici'', La Chimica e L'Industria, 66, 244-253, (1984).

29. G.Gardini, A.Berlin, '' I polimeri conduttori '', La Chimica e L' Industria, 73, 764-770, (1991).

30.       '' Handbook of Organic conductive Molecules and Polymers '' 4 Volume Set. Edited by H.S.Nalwa, Hitachi Research Laboratory , Japan ( 1997 ).

31 . V. J.Hearing, M.A.Lutzner '' Mammalian Melanosomal Proteins : Characterization on polyacrylamide Gel Electrophoresis '', Yale Journal of Biology and Medicine, 46, 553-559,  (1973 );T. Kushimoto, V. Basrur, J. Valencia, J. Matsunaga, W.D. Vieira, V.J. Ferrans, J. Muller, E. Appella, V.J. Hearing "A model for melanosome biogenesis based on the purification and analysis of early melanosomes" Proc. Natl. Acad. Sci. U. S. A. 98, 10698, (2001).

32 ( 49 )  A.Korner et al. J.Invest.Dermatol.75,192, 1980.

33. P.Aroca et al., BBA,1035, 266-275, (1990).

34 .  A.Palumbo, M.d' Ischia , G.Misuraca, L.De Martino, G.Prota '' A new dopachrome-rearranging from the ejected ink of the cuttlefish Sepia officinalis ''  Biochem.J. 299, 839, (1994) ; BBA, 925, 203, (1987) .

35. P.A.Riley, '' Melanin ''J.Biochem.Cell Biol., 29, 1235, 1997.

36.  S.Ito, BBA, 883, 155, (1986).

37. R.A.Nicolaus '' Biogenesi delle melanine ''Accademia Nazionale dei Lincei, (Conferenze), Biogenesi delle sostanze naturali, Roma 1964, 291-319. Vedi pag.317.

 R.A.Nicolaus '' Biogenesis of melanins ''  Rassegna di Medicina Sperimentale, Anno IX, Suppl. N° 1,   ( 1962 ). V.Idelson  Ed., Naples 1962.

37.bis   C.Lambert, J.N.Chacon, M.R.Chedekel, E.J.Land, P.A.Riley, A.Thompson, G.Truscott '' A pulse radiolisis investigation of the oxidation of indolic melanin precursors : Evidence for indolequinone and subsequent intermediates ''  BBA, 993, 12-20, ( 1989 )

38. G.A.Duff, J.E.Roberts, N.Foster, '' Analysis of the structure of synthetic and natural melanins by solid phase NMR '' Biochemistry 27, 7112-7116, 1988.

39. M.G.Peter, H.Foster, On the structure of eumelanins : identification of costitutional patterns by solid-state NMR spectroscopy, Angew. Chem. Int. Ed. English.28,741-743, 1989.

40. S. Aime, M. Fasano,  C.Croonbridge, '' Solid-state 13C NMR characterization of melanin free acids from biosynthetic and natural melanins.'' Gazz.Chim.Ital. 120, 663-664, 1990

41 Aime, S.Fasano, M., Terreno, E., Croonbridge, C.J. '' NMR studies of melanins: Characterization of a solubile melanin free acid from Sepia ink.''  Pigment Cell Res. 1991, 4, 216-221

42 Hervé, M., Hirshinger, J., Granger, P.,Gilard, P., Deflandre, A., Goetz, N.: A 13C solid-state NMR study of the structure and auto-oxidation process of natural and synthetic melanin. Biochim.Biophys. Acta 1994 1204, 19-27.

43. A.Pezzella, M.D' Ischia , A.Napolitano, A.Palumbo, G.Prota '' An integrated approach to the study of sepiomelanin:Evidence for a high proportion of degraded 5,6-dihydroxyindole-2-carboxylic acid units in the pigment backbone ''   Tetrahedron, 53, 8281-8286,  ( 1997 ) . 

44. A.Napolitano,A.Pezzella, G.Prota, R.Seraglia, P.Traldi  '' Structural analysis  of synthetic melanins from 5,6-Dihydroxyindole by Matrix - assisted Laser desorption / ionization Mass Spectrometry '' Rapid Comm. Mass Spectrom., 10, 468-472 ( 1996 ) ;   ‘’ A  reassessment of the structure of 5,6-Dihydroxyindole -2-carboxylic acid Melanins by Matrix-assisted Laser Desorption/ionization Mass Spectrometry ‘’ 10, 204-208 (1996).

45. A.Pezzella, A.Napolitano, M.d' Ischia , G.Prota, R.Seraglia, P.Traldi '' Identification of partially degraded oligomers of 5,6-Dihydroxyindole-2-carboxylic acid in Sepia melanin by Matrix-assisted Laser desorption/ionization Mass Spectrometry '' Rapid.Comm.Mass Spectrom. 11, 368-373, 1997

46. A.Bertazzo, C.Costa, G.Allegri, R.Serraglia, P.Traldi '' Biosynthesis of melanin from dopamine. An investigation of early oligomerization products ''  Rapid Comm. Mass Spectrom.  9, 634-640      ( 1995 ).

47. A.Bertazzo, C.Costa, G.Allegri, M.Schiavolin, D.Favretto, P.Traldi '' Enzymatic oligomerization of tyrosine  by tyrosinase and peroxidase studied by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry ''  Rapid Comm. Mass Spectrom, 13, 542-547,  ( 1999 ).

48. A. Korner, J.Pawelek, '' Mammalian tyrosinase catalyzes three reactions in the biosynthesis  of melanin '' Science, 217, 1163-1165,  ( 1982 ).

49. A.Korner, J.Pawelek,  '' Dopachrome conversion  : a possible control point in melanin biosynthesis '', J.Invest.Dermatol., 75, 192-195,  ( 1980 ).

 J.Pawelek, A.Korner, A.Bergstrom, J.Bologna ''New Regulators of Melanin biosynthesis  and the autodestruction  of melanoma cell '', Nature, 286, 617-619, ( 1980 ).

49 bis. P. Aroca, F. Solano, J.C. Garcia-Borron, J.A. Lozano "Specificity of dopachrome tautomerase and inhibition by carboxylate indoles. Considerations on the enzyme active site" Biochem. J. 277, 393, (1991).

50. B. Kagedal, P. Konradsson, T. Shibata, Y. Mishima "High-performance liquid-chromatographic analysis of dopachrome and dihydroxyphenylalanine" Anal. Biochem. 225, 264, (1995)

51.   P.Aroca, F.Solano, J.C Garcia-Borron, J.A.Lozano ''A new spectrophotometric assay for dopachrome  tautomerase ''J.Biol.Biophys.Methods, 21, 35-46  (1990)

52. F.Solano, C.Jimenez-CervantesJ.H.Martinez-Liarte, J.C.Garcia-Borron, J.R.Jara, J.A.Lozano '' Molecular Mechanism for catalysis by a new zinc-enzyme, dopachrome tautomerase '' Biochem.J., 313, 447-453,  ( 1996 ).

53. J.L.Pennock, J.M.Behnke, Q.D.Bickle, E.Devaney, R.K.Grencis, R.E.Isaac, G.W.P., Joshua, M.E.Selkirk, Y.Zhang, D.J.Meyer ''  Rapid purification and cheracterization of L-dopachrome-metyl ester tautomerase  ( macrophage-migration-inhibitory factor ) from Trichinella spiralis, Trichuris muris and Brugia pahangy ''  Biochem.J., 335, 495-498, ( 1998 ).

54. a. J. Pawelek " Is dopachrome tautomerase necessary to get DHICA from dopachrome? " Pigment. Cell Res. 7, 126, (1994)

b.F. Solano, J.C. Garcia-Borron, J.A. Lozano "Is dopachrome tautomerase necessary to get DHICA from dopachrome? ''  Pigment Cell Res. 7, 125, (1994)

c.S. Pavel "Dopachrome tautomerase is not essential for DHICA formation" Pigment Cell Res. 7, 123, (1994).

55. H. Sugimoto, M. Taniguchi, A. Nakagawa, I. Tanaka, M. Suzuki, J. Nishihira "Crystallization and preliminary X-ray analysis of human D-dopachrome tautomerase" J. Struct. Biol. 120, 105, (1997).

56. H. Sugimoto, M. Taniguchi, A. Nakagawa, I. Tanaka, M. Suzuki, J. Nishihira "Crystal Structure of human D-dopachrome tautomerase, a homologue of macrophage migration inhibitory factor, at 1,54 A resolution" Biochem. 38, 3268, (1999).

57. H. Yoshida, J. Nishihira, M. Suzuki, K. Hikichi "NMR characterization of physicochemical properties of rat D-dopachrome tautomerase" Biochem. Mol. Biol. Int. 42, 891, (1997).

58. G. Odh, A. Hindemith, A.M. Rosengren, E. Rosengren, H. Rorsman "Isolation of a new tautomerase monitored by the conversion of D-dopachrome to 5,6-dihydroxyindole" Biochem. Biophys. Res. Commun. 197, 619, (1993).

59. S.L.Jacques , D.J.McAuliffe '' The melanosome :Threshold temperature for explosive vaporization and internal absorption  coefficient during pulsed LASER irradiation ''   Photochem.Photobiol. 53, 769, ( 1991 ).

60. J.Borovansky, P. Hach, J.Duchon '' Melanosome  : an unusually resistant subcellular particle '' Cell Biology International Reports, Vol.1, 549-554 ( 1977 ) .

Melanosome are resistant to acid hydrolysis but they are easily degraded  by LASER or , as occur in vivo, by H2O2.

61. M.Bakunin, G.Dragotti '' Contributo alla conoscenza dei pigmenti melanici ''Rend. Real. Acc. Sci. Fis.Mat. Vol. X, 222, ( 1904 )

 62. a. R.A.Nicolaus,M.Piattelli, G.Narni, , '' The structure of sepiomelanin'' Tetrahedron, Letters 21, 14-17, (1959).

b. M.Piattelli, R.A.Nicolaus '' The structure of melanins and melanogenesis-I-The structure of melanin in Sepia '' Tetrahedron  15, 66-75, (1961).

c. M.Piattelli, E.Fattorusso,S.Magno, R.A.Nicolaus '' The structure of melanins and melanogenesis-II-Sepiomelanin and synthetic pigments '' Tetrahedron 18, 941-949, (1962).

d. M.Piattelli,E.Fattorusso, S.Magno,R.A.Nicolaus '' The structure of melanins and melanogenesis -III-The structure of sepiomelanin '' Tetrahedron, 19, 2061-2072 (1963)

e. R.A.Nicolaus, M.Piattelli, E.Fattorusso ''The structure of melanins and melanogenesis -IV-On some natural melanins '' Tetrahedron, 20, 1163-1172 (1964).

63. J.Medrano, D.Dudis ''Quasi-particles in polymeric conductors '' in Organic Superconductivity, pag.275. Ed. V.Z.Kresin, W.A.Little, Plenum Press, New York ,  (1990 )

64. R.Nicolaus '' Sugli acidi pirrolcarbonici.Nota I. Acido 2,3,5-pirroltricarbonico '' Gazz.Chim.Ital. 83, 239-251, 1953

65. R.Nicolaus,G.Oriente ''Sugli acidi pirrolcarbonici : acido 2,3,4,5 pirroltetracarbonico '' Gazz.Chim.Ital. 84, 230-241, 1954

66. E.Fattorusso, L.Minale, S.De Stefano, G.Cimino, R.A.Nicolaus '' Struttura e biogenesi delle feomelanne.Nota V.-Sulla struttura della gallofeomelanina1 '' Gazz.Chim.Ital. 98, 1443-1463, 1968.

67. S.Ito, K.Fujita '' Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical dgradation and liquid chromatography '' Analytical Biochemistry  144, 527-536, 1985

68. P.Manini, M.D' Ischia , M.Milosa, G.Prota '' Acid-promoted competing pathways in the oxidative polymerization of 5,6-Dihydroxyindoles and related compounds : straightforward cyclotrimerization routes to diindolocarbazole  derivatives '' J.Org.Chem, 63, 7002-7008, 1998.

69 .a G.A. Swan "Chemical Structure of Melanins" Annals of the New York Academy of Sciences Vol. 100, 1005 (1963)

b.F. Binns, G.A. Swan "Oxidation of some Synthetic Melanins" Chemistry and Industry 396 (1957)

c.      G.A. Swan "Some studies on the formation and structure of melanins" Rendiconti Accademia   Scienze Fisiche Matematiche, Vol. XXXI (1964)

Studies are described of the formation of melanins in vitro  enzymically and by oxidation from dopa and from dopammine.When these precursors were labelled with deuterium  in the alfa and beta positions of the side chain, and then converted into melanins large retention of deuterium in melanin was observed.This means that melanins are not DHI-melanins.

d.S.N. Mishra, G.A. Swan "Studies related to the chemistry of Melanins. -Part II- Synthesis of 5,6-Dihydroxyindoline" Soc. 1424 (1967).

e.S.N. Mishra, G.A. Swan "Studies related to the chemistry of Melanins -Part V- Investigations on the Specific Deuteriation of 5,6-Dihydroxyindoline and 5,6-Dihydroxyindole" Soc. 1431 (1967).

f.F. Binns, J.A. King, A. Percival, N.C. Robson, G.A. Swan " Studies related to the Chemistry of Melanins -Part IX- Syntheses of Specifically Deuteriated 3,4-Dihydroxyphenethylamines and

 3,4-Dihydroxyphenylalanines " Soc. 1134 (1970).

g. F. Binns, J.A. King, S.N. Mishra, A. Percival, N.C. Robson, G.A. Swan, A. Waggott,"Studies related to the Chemistry of Melanins -Part. XIII- Studies on the structure of dopamine-melanin" Soc. 2062 (1970).

h. G.A. Swan "Structure Chemistry and Biosynthesis of Melanins in Fort. Chem. Org. Natur. Vol. 31, 522, (1974) Springer-Verlag,  Wien 1974

The experiments show that 20% of the polymer units are formed by DOPA and cyclodopa units either in the autoxidative or enzymatic process. This value was cofirmed by (58) by NMR.

i. G.A.Swan '' Current knowledge of Melanin Structure '' In Pigment Cell,Vol. 1, Eds.V.J     McGovern,P.Russel. S.Karger, Sydney , (1973 ), pag.151.

If the Raper Scheme were correct it would be expected that the same melanin would be       obtained from tyrosine, DOPA, dopammine,DHI. .Although these melanins have the same radical-  polarone system, they are,in part, chemically different.

l. J.A. King, A. Percival, N.C. Robson, G.A. Swan "Studies related to the Chemistry of Melanins -XI- The distribution of polymeric linkages in dopamelanin" Soc. 1418 (1970).

m. G.W. Kirby, L. Ogunkoya " Structure of melanin derived from 3,4 -dihydroxy- 1 - (14C, 3H) -phenylalanine by oxidation with tyrosinase " Soc. Chem. Comm. 21 546 (1965).

70.  V.J. Hearing, T.M. Ekol, P.M. Montague, J.M. Nicholson " Mammalian Tyrosinase Stoichiometry and measurement of reaction product " BBA  611, 251 (1980).

In this important paper intermediates involved in the conversion of tyrosine or DOPA into melanin were studied by a variety of enzyme assays:The data indicate that uncyclised  or carboxylated units are, in agreement with recent results 2004, not incorporated into the polymer in vitro and probably also in vivo.

The authors reported that there is  no incorporation of DOPA, dopachrome,    leucodopachrome   (cyclodopa), DHICA into dopamelanin..In other words in  melanins the carboxylic  present is formed by fission of the benzenoid part of the molecule.

See also Allegri (138). Peter (139) 

70a .  M.Seiji, T.B.Fitzpatrick, R.T.Simpson, M.S.C.Birbeck  ''Chemical composition and terminology of specialized organelles ( melanosomes and melanin granules ) in mammalian melanocytes '' Nature 197, 1082-1084, 1963.

71. M.Seiji, K.Shimao, M.S.C.Birbeck, T.B.Fitzpatrick  ''Subcellular localization of melanin biosynthesis '' in The Pigment Cell : Molecular, Biological and Chemical Aspects, Ed. P.Riley, J.C.Fortner, New York Academy of Sciences, 100, 497 , 1963.

72. A.Pezzella, A.Napolitano, M.D' Ischia , G.Prota,R.Seraglia, P.Traldi '' Identification partially degraded oligomers of 5,6-dihydroxyindole-2-carboxylic acid in Sepia melanin by matrix-assisted LASER desorption/ionization mass spectrometry '' Rapid Comm.Mass Spectr. 11, 368-372, 1997.

73. H.Wyler, J.Chiovini '' Die Synthese von Cyclodopa ( leucodopachrome) '' Helv.Chim.Acta 51, 1476-1482, 1968.

74.A.Aroca, F.Solano, C.Solinas, J.C.Garcia-Borron, J.A.Lozano, Eur.J.Biochem. 208, 155-163, 1992

75. H.Wyler, '' The dopachrome-dihydroxyindole '' in   Melanogenesis.Its chemistry as a therapeutic sraregy in melanoma. Manchester Meeting   1991

76. B.J.R.Nicolaus, R.A.Nicolaus '' Stories of melanins  (BCM) ''  Atti Accademia Pontaniana Vol. LI, Napoli ,26 Giugno , 2003.

77. a. K.Hempel '' Investigation on the structure of melanin in malignant melanoma with 3H- and 14C-dopa labeled at different position '' . Structure and control of the melanocyte. Eds. G.Della Porta, O.Muhlbock, SV Berlin 1966, pp. 162-175.

      b. K.Hempel, '' Uber biosynthese und structur des tierischen melanins '' Z.Naturforschung 22, 173-178 ( 1967 ) ; K.Hempel, W.Erb, Z.Zellforschung, 58, 125-129, ( 1962). ; K.Hempel,M.Dennel '' Meeting of Gesell.Physiol.Chem. Wien 1962 ; K.Hempel, '' Sixth  Int. Pigment Cell Conference '' Sofia ( Bulgaria ), May 25-29, ( 1965 ).

Naples March 2003.

78. P.N.Patil '' Some factors which affect the ocular drug responses '' TIPS,May 1984 pag.201

79. M.S.Blois '' On Chloropromazine Binding in vivo '' J.Invest.Dermatol. 45, 475,  ( 1965 )

80. Letteratura riportata ( pag.187-216 ) in R.A.Nicolaus '' Divagazioni sulla struttura a banda del colore in natura : il nero. '' Rend.Acc.Sci.Fis.Mat., Vol. LXIV, 145-216, ( 1977 ).

81. H.Rorsman '' Binding of simple chemicals in melanin producing cell '' in Research in Organic, Biological, and Medicine ChemistryVol.3,Part II, pag 655, NHPC, Amsterdam (1972).

82. U.  Mårs  “Melanogenesis as the basis for melanoma targetin”  Acta Universitatis Upsaliensis. 188, 1-48, Uppsala,  (1998).

83 a. Blarzino C., Mosca L., Foppoli C., Coccia R., De Marco C. and Rosei M. A. (1999) Lipoxygenase/H2O2-catalyzed oxidation of dihydroxyindoles: synthesis of melanin pigments and study of their antioxidant properties. Free Radic. Biol. Med. 26, 446-451.

b. Mosca L., De Marco C., Fontana M. and Rosei M. A. (1999) Fluorescence properties of melanins form opioid peptides. Arch. Biochem. Biophys. 371, 63-69.

c. Rosei M. A., Coccia R., Foppoli C., Blarzino C., Cini C. and Schininà M. E. (2000) Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors. Biochim. Biophys. Acta 1478, 19-29.

d. Mosca L., De Marco C., Visioli F. and Cannella C. (2000) Enzymatic assay for the determination of olive oil polyphenol content: assay conditions and validation of the method. J. Agric. Food Chem. 48, 297-301.

e. Coccia R., Foppoli C., Blarzino C., De Marco C. and Rosei M. A. (2001) Interaction of enkephalin derivatives with reactive oxygen species. Biochim. Biophys. Acta 1525, 43-49.

84. S.Vogliardi,G.Allegri, A.Bertazzo, C.V.Costa, R.Seraglia, P.Traldi  '' An investigation on the role of 5-hydroxytryptophane in the biosynthesis of melanins  '' J.Mass Spectrom., 37, 1292-1296 , (2002).

85. K.Wakamatsu, S.Ito '' Advanced chemical methods in melanin determination '' Pigment Cell Research, 15, 174-183, (2002)

 

 

RECENT PAPERS TO THE ATTENTION OF BCM EXPERTS and students

85.a.

Bhavin B.Adhyaru, Novruz G. Akhmedov, Alan R.Katrizky, and Clifford R.Bowers   ‘’Solid-state cross-polarization magic angle spinning 13C and 15N NMR characterization of Sepia melanin, Sepia melanin free acid and Human hair melanin in comparison with several model compounds ‘’ , Magn., Reson. Chem., 2003, 43 : 466-474              

  One of the most interesting paper published in the field. Comparative examination of sepia  melanin with DHICA-melanin and DHI-melanin could be interesting…..

86.    E.J.Land, C.A. P.A.Riley '' Pulse radiolysis studies of ortho-quinone chemistry relevant to melanogenesis '' J.Photochem.Photobiol. B, 64, 123-135,  (2001).

87.    A.Slominsk, J.Wortsman, J.A. Carlson, L.Y.Matsuoka, C.M.Balch, M.C.Mihm '' Malignant Melanoma '' Arch.Pathol.Lab.Med., 125, 1295-1306, (2001).

88.    C.Jimenez-Cervantes, M.Martinez-Esparza, C.Perez, N.Daum, F.Solano, J.C.Garcia-Borron '' Inhibition of melanogenesis in response to oxidative stress : transient downregulation of melanocyte differentiation marker and possible involvement of microphthalmia trnscription factor '' J.Cell Sci. 114, 2335-2344, (2001).

89. A.Bertazzo, S.Vogliardi, D.Favretto, C.V.Costa, G.Allegri, P.Traldi '' Melanogenesis by tyrosinase action on 3,4-dihydroxyphenylalanine (DOPA) in the presence of polyethylene glycol :   a matrix-assisted LASER desorption/ionozation mass spectometric investigation '' Rapid Comm.Mass Spectrom. 15, 1061-1067, (2001).

90.  D.A.Samuelson, P.A.Lewis, E.MacKay, R.D.Whitley '' The influence of aging and low nutrition on the choroid in the pig : II. The melanosome. ''  Ve. Ophthalmol, 2,35-45, ( 1999 )

91.   Y.Mishima '' New era of cell re-discovery led to the control of melanogenesis/melanoma  ;  a scientific journey into terra incognita '' Pigment Cell Res., 14, 47-70, ( 2001 ).

92.   D.J.Tobin,R.Paus '' Graying :  gerontobiology of the hair follicle pigmentary unit  ''.Exp.Gerontol. 36, 29-54 ( 2001 ). 

93.  Y.Funasaka, M.Komoto, M.Ichihashi '' Depigmenting effect of alpha-tocopheryl ferulate on normal human melanocytes '' Pigment Cell Res. 13, 170-174, ( 2000 ).

94.  K.Jimbow, C.Hua, P.F.Gomez, K.Hirosaki,H.Matsusaka, H.Y.Jin, T.Yamashita '' Intracellular vesicular trafficking of tyrosinase protein in eu-and pheomelanosome biogenesis '' Pigment Cell Res. 13/8, 110-117, (2000).

95.  Y.Mishima, H.Kondoh '' Dual control of melanogenesis and melanoma growth : from molecular to clinical level and the reverse ''  Pigment Cell Res.13, 10-22, (2000).

96.  E.J.Land, P.A.Riley  ''Spontaneous reactions of dopaquinone and the balance between the eumelanic and pheomelanic pathways '' Pigment Cell Res., 13, 273-277, (2000).

97.  M.Seiberg, C.Paine, E.Sharlow, P.Andrade-Gordon, M.Costanzo, S.S.Shapiro '' Inhibition of melanosome transfer results in skin lightening '' J.Invest.Dermatol., 115, 162-167, (2000)

98.  A.Camacho-Hubner, F.Beermann  ''  Cellular and molecular features of mammalian pigmentation-tyrosinase and TRP ''  Pathol.Biol.(Paris) 48, 577-583.

99.    C.Lambert, J.N.Chacon, M.R.Chedekel, E.J.Land, P.A.Riley, A.Thompson, T.G.Truscott "A pulse radiolysis investigation of the oxidation of indolic melanin precursors: evidence for indolequinones and subsequent intermediates "BBA, 993, 12-20, (1989).

100.   D.G.Graham, P.W.Jeffs, "The role of 2,4,5-Trihydroxyphenylalanine in Melanin biosynthesis" J.Biol.Chem. 252, 5729-5734, (1977).  

101.   S. Ito " The IFPCS presidential lecture: a chemist's view of melanogenesis" PCR 16, 230-236, (2003).

102  R.Seraglia, P.Traldi, G.Elli, A.Bertazzo, C.Costa, G.Allegri     Laser desorption ionization Mass Spectrometry in the study of natural and synthetic melanins. I-Tyrosine Melanins.  “, Biol.Mass Spectrometry,  22, 687-697, (1993).

 

 Pezzella, Di Vogna, G. Prota “Synthesis of optically active tetrameric melanin intermediates by oxidation of the melanogenic precursor 5,6-dihydroxyindole-2-carboxylic acid under biomimetic conditions” Tetrahedron asymmetry  14, 1133-1140, (2003).

ALLOMELANIN and PLANT-MELANIN

 

103.  F.Solano, D.Hernandez-Romero, D.Lopez-Serrano, A.Sanchez-Amat ‘’ Polyphenol oxidase systems and pigment formation in Raistonia solanacearum ‘’  Pigment Cell Research, 16,594, 2003

Substantia 8.doc

104a1.  Tsuji G, Sugahara T, Fujii I, Mori Y, Ebizuka Y, Shiraishi T, Kubo Y.

 Evidence for involvement of two naphthol reductases in the first reduction step

of melanin biosynthesis pathway of Colletotrichum lagenarium.

Mycol Res. 107, 854-60, 2003..

 

104a2. Solano F, Hernandez-Romero D, Lopez-Serrano D, Sanchez-Amat A

 Polyphenol oxidase systems and pigment formation in Ralstonia

solanacearum.

Pigment Cell Res.16, 594, 2003.

 

104a3.  Haslam E.

 Thoughts on thearubigins.

Phytochemistry. 2003 Sep+ADs-64(1):61-73. Review.

 

104a4.  Doss RP, Deisenhofer J, Krug von Nidda HA, Soeldner AH, McGuire RP.

 Melanin in the extracellular matrix of germlings of Botrytis cinerea.

Phytochemistry. 2003 Jul+ADs-63(6):687-91.

 

104a5.  Ohguchi K, Tanaka T, Iliya I, Ito T, Iinuma M, Matsumoto K, Akao Y, Nozawa

  Gnetol as a potent tyrosinase inhibitor from genus Gnetum.

Biosci Biotechnol Biochem. 2003 Mar+ADs-67(3):663-5.

 

104a6  Lanisnik Rizner T, Wheeler MH.

 Melanin biosynthesis in the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus).

Can J Microbiol. 2003 Feb+ADs-49(2):110-9.

 

104a7.  Lee KT, Lee KS, Jeong JH, Jo BK, Heo MY, Kim HP.

 Inhibitory effects of Ramulus mori extracts on melanogenesis.

J Cosmet Sci. 2003 Mar-Apr+ADs-54(2):133-42.

 

104a8.  Johnson SM, Doherty SJ, Croy RR.

 Biphasic superoxide generation in potato tubers. A self-amplifying response to stress.

Plant Physiol. 2003 Mar+ADs-131(3):1440-9.

 

104a9.  Langfelder K, Streibel M, Jahn B, Haase G, Brakhage AA.

 Biosynthesis of fungal melanins and their importance for human pathogenic fungi.

Fungal Genet Biol. 2003 Mar+ADs-38(2):143-58. Review.

 

104a10.  Schempp CM, Winghofer B, Muller K, Schulte-Monting J, Mannel M, Schopf E,Simon JC.

 Effect of oral administration of Hypericum perforatum extract ( St. John's Wort)on skin erythema and pigmentation induced by UVB, UVA, visible light and solarsimulated radiation.

Phytother Res. 2003 Feb+ADs-17(2):141-6.

 

104a11.  Yamashita M, Ohta N, Shimizu T, Matsumoto K, Matsuura Y, Kawasaki I, TanakaT, Maezaki N, Ohta S.

 First total synthesis of ()-Linderol A, a tricyclic hexahydrodibenzofuran

constituent of Lindera umbellata bark, with potent inhibitory activity on

melanin biosynthesis of cultured B-16 melanoma cells.

J Org Chem. 2003 Feb 21+ADs-68(4):1216-24.

 

104a12.  Yin PY, Lu MS, Kong QS, Rong R, Liu G.

 +AFs-Structure characterization of melanin in black sesame by GC/MS+AF0

Se Pu. 19, :268-9, 2001

 

104a13.  Cho SM, Kwon YM, Lee JH, Yon KH, Lee MW.

 Melanogenesis inhibitory activities of diarylheptanoids from Alnus hirsuta

Turcz in B16 mouse melanoma cell.

Arch Pharm Res. 2002 Dec+ADs-25(6):885-8.

 

104a14.  Hung YC, Sava VM, Blagodarsky VA , Hong MY, Huang GS.

 Protection of tea melanin on hydrazine-induced liver injury.

Life Sci. 2003 Jan 17+ADs-72(9):1061-71.

 

104a15.  Oh H, Mun YJ, Im SJ, Lee SY, Song HJ, Lee HS, Woo WH.

 Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on

tyrosinase activity and melanin synthesis of B16/F10 melanoma cells.

Planta Med. 2002 Sep+ADs-68(9):832-3.

 

104a16.  Jones K, Hughes J, Hong M, Jia Q, Orndorff S.

 Modulation of melanogenesis by aloesin: a competitive inhibitor of tyrosinase.

Pigment Cell Res. 2002 Oct+ADs-15(5):335-40.

 

104a17.  Lee SH, Choi SY, Kim H, Hwang JS, Lee BG, Gao JJ, Kim SY.

 Mulberroside F isolated from the leaves of Morus alba inhibits melanin

biosynthesis.

Biol Pharm Bull. 2002 Aug+ADs-25(8):1045-8.

 

104a18.  Hermanns JF, Petit L, Pierard-Franchimont C, Paquet P, Pierard GE.

 Assessment of topical hypopigmenting agents on solar lentigines of Asian women.

Dermatology. 2002+ADs-204(4):281-6.

 

104a19.  El Bassam S, Benhamou N, Carisse O.

 The role of melanin in the antagonistic interaction between the apple scab

pathogen Venturia inaequalis and Microsphaeropsis ochracea.

Can J Microbiol. 2002 Apr+ADs-48(4):349-58.

 

104a20. Castro-Sowinski S, Martinez-Drets G, Okon Y.

 Laccase activity in melanin-producing strains of Sinorhizobium meliloti.

FEMS Microbiol Lett. 2002 Mar 19+ADs-209(1):119-25.

 

104a21.  Capasso R, De Martino A, Arienzo M.

 Recovery and characterization of the metal polymeric organic fraction

(polymerin) from olive oil mill wastewaters.

J Agric Food Chem. 2002 May 8+ADs-50(10):2846-55.

 

104a22.  Carzaniga R, Fiocco D, Bowyer P, O'Connell RJ.

 Localization of melanin in conidia of Alternaria alternata using phage displayantibodies.

Mol Plant Microbe Interact. 2002 Mar+ADs-15(3):216-24.

 

104a23  Shimizu K, Kondo R, Sakai K, Takeda N, Nagahata T.

 The skin-lightening effects of artocarpin on UVB-induced pigmentation.

Planta Med. 2002 Jan+ADs-68(1):79-81.

 

104a24.  Cabanes J, Chazarra S, Garcia-Carmona F.

 Tyrosinase kinetics: a semi-quantitative model of the mechanism of oxidation ofmonohydric and dihydric phenolic substrates--reply.

J Theor Biol. 2002 Jan 21+ADs-214(2):321-8. No abstract available.

 

104a25.  Hung YC, Sava VM, Juang CL, Yeh T, Shen WC, Huang GS.

 Gastrointestinal enhancement of MRI with melanin derived from tea leaves (Theasinensis Linn.).

J Ethnopharmacol. 2002 Jan+ADs-79(1):75-9.

 

104a26.  Jordan DB, Basarab GS, Liao DI, Johnson WM, Winzenberg KN, Winkler DA.

 Structure-based design of inhibitors of the rice blast fungal enzyme

trihydroxynaphthalene reductase.

J Mol Graph Model. 2001+ADs-19(5):434-47, 470-1.

 

104a27.  Arend J, Warzecha H, Hefner T, Stockigt J.

 Utilizing genetically engineered bacteria to produce plant-specific glucosides.

Biotechnol Bioeng. 2001 Sep+ADs-76(2):126-31.

 

104a28.  West TP , Strohfus B.

 Polysaccharide production by a reduced pigmentation mutant of Aureobasidiumpullulans NYS-1.

Lett Appl Microbiol. 2001 Aug+ADs-33(2):169-72.

 

104a29. Pawelek J.M.

 Approaches to increasing skin melanin with MSH analogs and synthetic melanins.

Pigment Cell Res. 2001 Jun+ADs-14(3):155-60. Review.

 

104a30.  Yamashita M, Ohta N, Kawasaki I, Ohta S.

 The first total synthesis of ()-linderol A, a tricyclic

hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent

inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells.

Org Lett. 2001 May 3+ADs-3(9):1359-62.

 

104a31.  Gomez-Cordoves C, Bartolome B, Vieira W, Virador VM.

 Effects of wine phenolics and sorghum tannins on tyrosinase activity and growth

of melanoma cells.

J Agric Food Chem. 2001 Mar+ADs-49(3):1620-4.

 

104a32.  el-Sayed el-SA.

 Production of thaxtomin a by two species of Streptomyces causing potato scab.

Acta Microbiol Immunol Hung. 2001+ADs-48(1):67-79.

 

104a33.  Hermanns JF, Petit L, Martalo O, Pierard-Franchimont C, Cauwenbergh G,

Pierard GE.

 Unraveling the patterns of subclinical pheomelanin-enriched facial

hyperpigmentation: effect of depigmenting agents.

Dermatology. 2000+ADs-201(2):118-22.

 

104a34.  Jacobson ES.

 Pathogenic roles for fungal melanins.

Clin Microbiol Rev. 2000 Oct+ADs-13(4):708-17. Review.

 

104a35.  Castro S, Carrera I, Martinez-Drets G.

 Methods to evaluate nodulation competitiveness between Sinorhizobium meliloti

strains using melanin production as a marker.

J Microbiol Methods. 2000 Jul+ADs-41(2):173-7.

 

104a36.  Kamei H, Koide T, Kojima T, Hasegawa M, Umeda T.

 Suppression of growth of cultured malignant cells by allomelanins,

plant-produced melanins.

Cancer Biother Radiopharm. 1997 Feb+ADs-12(1):47-9.

 

104a37.   La Porta CA.

 nPKCdelta a new therapeutic marker for melanoma metastasis? (Review).

Int J Mol Med. 2000 May+ADs-5(5):467-71. Review.

 

104a38.  Liao DI, Basarab GS, Gatenby AA, Jordan DB.

 Selection of a potent inhibitor of trihydroxynaphthalene reductase by sorting

disease control data.

Bioorg Med Chem Lett. 2000 Mar 6+ADs-10(5):491-4.

 

104a39.Lapina VA, Sheshko PM, Pankovets EA, Dontsov AE.

 Phytosorbent prepared from sunflower seed husks prevents mercuric chlorideaccumulation in kidney and muscle of adult rabbits.

Arch Environ Health. 2000 Jan-Feb+ADs-55(1):48-50.

 

104a40  Sugumaran M, Nellaiappan K.

 Characterization of a new phenoloxidase inhibitor from the cuticle of Manducasexta.

Biochem Biophys Res Commun. 2000 Feb 16+ADs-268(2):379-83.

 

104a41.  Aquaron R.

 Tradition of basic and applied pigment cell research in Marseilles .

Cell Mol Biol (Noisy-le-grand). 1999 Nov+ADs-45(7):877-82. Review.

 

104a42.  Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M,Shapiro SS.

 The protease-activated receptor 2 regulates pigmentation via

keratinocyte-melanocyte interactions.

Exp Cell Res. 2000 Jan 10+ADs-254(1):25-32.

 

104a43.  Cadugo MA, Chua MG, Feliciano MA, Jimenez FC Jr, Uy HG.

 A preliminary clinical study on the oral lesions among the Dumagats.

J Philipp Dent Assoc. 1998 Sep-Nov+ADs-50(2):36-42.

 

104a44.  Kubo I, Kinst-Hori I.

 Flavonols from saffron flower: tyrosinase inhibitory activity and inhibition

mechanism.

J Agric Food Chem. 1999 Oct+ADs-47(10):4121-5.

 

104a45.  No JK, Soung DY, Kim YJ, Shim KH, Jun YS, Rhee SH, Yokozawa T, Chung HY.

 Inhibition of tyrosinase by green tea components.

Life Sci. 1999+ADs-65(21):PL241-6.

 

104a46.   Lin Z, Hoult JR, Bennett DC, Raman A.

Stimulation of mouse melanocyte proliferation by Piper nigrum fruit extract and its main alkaloid, piperine.

Planta Med. 1999 Oct+ADs-65(7):600-3.

 

194a47.  Lin ZX, Hoult JR, Raman A.

 Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo.

J Ethnopharmacol. 1999 Aug+ADs-66(2):141-50.

 

104a48. Edens WA , Goins TQ, Dooley D, Henson JM.

 Purification and characterization of a secreted laccase of Gaeumannomyces graminis var. tritici.

Appl Environ Microbiol. 1999 Jul+ADs-65(7):3071-4.

 

104a49. Rios M, Habecker B, Sasaoka T, Eisenhofer G, Tian H, Landis S, Chikaraishi D, Roffler-Tarlov S.

 Catecholamine synthesis is mediated by tyrosinase in the absence of tyrosine

hydroxylase.

J Neurosci. 1999 May 1+ADs-19(9):3519-26.

 

104a50.  Liao YL, Chiang YC, Tsai TF, Lee RF, Chan YC, Hsiao CH.

 Contact leukomelanosis induced by the leaves of Piper betle L. (Piperaceae): aclinical and histopathologic survey.

J Am Acad Dermatol. 1999 Apr+ADs-40(4):583-9.

 

104a51.   Xu Y, Stokes AH, Roskoski R Jr, Vrana KE.

 Dopamine, in the presence of tyrosinase, covalently modifies and inactivatestyrosine hydroxylase.

J Neurosci Res. 1998 Dec 1+ADs-54(5):691-7.

 

104a52.  Lichter A, Mills D.

 Control of pigmentation of Ustilago hordei: the effect of pH, thiamine, andinvolvement of the cAMP cascade.

Fungal Genet Biol. 1998 Oct+ADs-25(1):63-74.

 

104a53.  Money NP, Caesar-TonThat TC, Frederick B, Henson JM.

 Melanin synthesis is associated with changes in hyphopodial turgor,

permeability, and wall rigidity in gaeumannomyces graminis var. graminis.

Fungal Genet Biol. 1998 Jun-Jul+ADs-24(1-2):240-51.

 

104a54.  Avramidis N, Kourounakis A, Hadjipetrou L, Senchuk V.

 Anti-inflammatory and immunomodulating properties of grape melanin. Inhibitoryeffects on paw edema and adjuvant induced disease.

Arzneimittelforschung. 1998 Jul+ADs-48(7):764-71.

 

104a55.  Shimizu K, Kondo R, Sakai K, Lee SH, Sato H.

 The inhibitory components from Artocarpus incisus on melanin biosynthesis.

Planta Med. 1998 Jun+ADs-64(5):408-12.

 

104a56.  Clarys P, Barel A.

 Efficacy of topical treatment of pigmentation skin disorders with plant

hydroquinone glucosides as assessed by quantitative color analysis.

J Dermatol. 1998 Jun+ADs-25(6):412-4.

 

 

 

104a57.  Riley PA.

 Melanin.

Int J Biochem Cell Biol. 29, 1235-9, 1997. Review.

 

104a58  Kahn V, Ben-Shalom N.

 Effect of maltol on the oxidation of DL-DOPA, dopamine, N-acetyldopamine

(NADA), and norepinephrine by mushroom tyrosinase.

Pigment Cell Res. 10,139-49, 1997.

 

105. V.M.Sava, Y.C.Hung, V.A.Blagodarsky, M.Y.Hong, G.S.Huang  ?? The liver-protecting activity of melanin-like pigment derived from black tea ‘’ Food Research International 36, 505-511, 2003

106.  S.Tian, J.Garcia-Rivera, B.Yan, A.Casadevall, R.E.Stark  ‘’ Unlocking the molecular structure of fungal melanine using 13C biosynthetic labeling and solid-state  NMR ‘’  Biochemistry, 15, 8105-8109, 2003.

107. R.Morris-Jones, S.Youngchim, B.L.Gomez, P.Aisen, R.J.Hay, J.D.Nosanchuk, A.Casadevall, A.J.Hamilton  ‘’ Synthesis of melanin-like pigments by Sporothrix schenckii  in vitro and during mammalian infection  ‘’  Infect.Immun., 71, 4026-4033, 2003

108. J.Matsubara, J.Takahashi, K.Ikeda, Y.Shimizu, S.Matsui  ‘’  The effects of humic substances on the intake of micro-organic pollutans into the acquatic biota ‘’  Water Sci.Technol. 47, 117-124, 2003

109. T.L.Rizner, M.H.Wheeler ‘’ Melanin biosynthesis in the fungus Curvularia lunata  (teleomorph : Cochliobulus lunatus  ) ‘’ Canadian Journal of Microbiology 49,  X, 2003.

 

 

BINDING

 

 

110. C.R.Roberts, J.C.Briggs, D.G.Wilkins, D.E.Rollins ‘’ Cocaine,Benzoylecgonine, Amphetamine, and N-Acetylamphetamine Binding to melanin Subtypes ‘’ Journal of Analytical Toxicology, 27, 142-144, 2003.

111. R.Kronstrand, J.Ahiner, N.Dizdar, G.Larson ‘’ Quantitative analysis of desmethylselegiline, methamphetamine, and amphetamine in hair and plasma from Parkinson patients on long-term selegiline medication ‘’ Journal of Analytical Toxicology, 27,135-143, 2003.

112. J.Oikos ‘’ Melanins,metals, and mate quality ‘’ Oikos, 102, 402-406, 2003.

113. Y.Liu, V.Kempf, A.Samokhvalov, J.D.Simom ‘’ Metal ions complexation with melanin ‘’  Pigment Cell Research 16, 597, 2003.

Substantia 9.doc

 

113a.  Hung YC, Sava V, Hong MY, Huang GS.

 Inhibitory effects on phospholipase A2 and antivenin activity of melanin

extracted from Thea sinensis Linn.

Life Sci. 74, 2037-47, 2004

 

113a1.  Rollins DE, Wilkins DG, Krueger GG, Augsburger MP, Mizuno A, O'Neal C,Borges CR, Slawson MH.

 The effect of hair color on the incorporation of codeine into human hair.

J Anal Toxicol. 27, 545-51, 2003.

 

113a2.  Koeberle MJ, Hughes PM, Skellern GG, Wilson CG.

 Binding of memantine to melanin: influence of type of melanin and

characteristics.

Pharm Res. 2003 ,20, 1702-9.

 

113a3.  Mieczkowski T.

 Assessing the potential of a "color effect" for hair analysis of

11-nor-9-carboxy-delta(9)-tetrahydrocannabinol: analysis of a large sample of hair specimens.

Life Sci. 2003 74, 463-9.

 

113a4.  Healy E, Robinson SJ, Dixon SV.

 IL-13 Alpha-melanocyte stimulating hormone; a matter of life and death.

Pigment Cell Res. 2003, 16, 577.

 

 

113a5.  Kuroda TS, Ariga H, Fukuda M.

 The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes.

Mol Cell Biol. 2003 , 23, 5245-55.

 

113a6. Farmer PJ, Gidanian S, Shahandeh B, Di Bilio AJ, Tohidian N, Meyskens FL Jr.

 

 Melanin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals on melanoma viability.

Pigment Cell Res. 2003, 16, 273-9.

 

113a6.  Kim DH, Hwang JS, Baek HS, Kim KJ, Lee BG, Chang I, Kang HH, Lee OS.

 Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent.

Chem Pharm Bull ( Tokyo ). 2003, 51, 113-6.

 

 

 

113a7.  Korotkova TM, Sergeeva OA, Eriksson KS , Haas HL, Brown RE.

 Excitation of ventral tegmental area dopaminergic and nondopaminergic neurons by orexins/hypocretins.

J Neurosci. 2003, 23, 7-11.

 

 

 

113a8.  Audinot V, Lahaye C, Suply T, Rovere-Jovene C, Rodriguez M, Nicolas JP,Beauverger P, Cardinaud B, Galizzi JP, Fauchere JL, Nahon JL, Boutin JA.

 SVK14 cells express an MCH binding site different from the MCH1 or MCH2receptor.

Biochem Biophys Res Commun. 2002 , 295, 841-8.

 

113a9. Wrzesniok D, Buszman E, Karna E, Nawrat P, Palka J.

 Melanin potentiates gentamicin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

Eur J Pharmacol. 2002 , 446, 7-13.

 

113a10. Jequier E.

 Leptin signaling, adiposity, and energy balance.

Ann N Y Acad Sci. 2002, 967, 379-88. Review.

 

113a11.  Erkert RS, Macallister CG.

 Isoxsuprine hydrochloride in the horse: a review.

J Vet Pharmacol Ther. 2002, 25, 81-7. Review.

 

113a12. Wittkopp PJ, True JR, Carroll SB.

 Reciprocal functions of the Drosophila yellow and ebony proteins in the

development and evolution of pigment patterns.

Development. 2002, 129, 1849-58.

 

113a13.  Shimizu K, Kondo R, Sakai K, Takeda N, Nagahata T.

 The skin-lightening effects of artocarpin on UVB-induced pigmentation.

Planta Med. 2002, 68, 79-81.

 

113a14.  Yoshida M, Hirotsu S, Nakahara M, Uchiwa H, Tomita Y.

 Histamine is involved in ultraviolet B-induced pigmentation of guinea pig skin.

J Invest Dermatol. 2002, 118, 255-60.

 

113a15.  Voisey J, van Daal A.

 Agouti: from mouse to man, from skin to fat.

Pigment Cell Res. 2002, 15, 10-8. Review.

 

113a16.  Varas M, Perez M, Ramirez O, de Barioglio SR.

 Melanin concentrating hormone increase hippocampal synaptic transmission in the rat.

Peptides. 2002 , 23, 151-5.

 

113a17. Mani I, Sharma V, Tamboli I, Raman G.

 Interaction of melanin with proteins--the importance of an acidic

intramelanosomal pH.

Pigment Cell Res. 2001, 14,170-9.

 

113a18. Surazynski A, Palka J, Wrzesniok D, Buszman E, KaczmarczykP.

 Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis inhuman skin fibroblasts.

Eur J Pharmacol. 2001, 419, 139-45.

 

 

113a19  Smith-Thomas LC, Richardson PS, Rennie IG, Palmer I, Boulton M, Sheridan C, MacNeil S.

 Influence of pigment content, intracellular calcium and cyclic AMP on the ability of human retinal pigment epithelial cells to contract collagen gels.

Curr Eye Res. 2000, 21, 518-29.

 

113a20.  Bhardwaj R, Hadley ME, Dorr RT, Dvorakova K, Brooks C, Blanchard J.

 Pharmacologic response of a controlled-release PLGA formulation for the

alpha-melanocyte stimulating hormone analog, Melanotan-I.

Pharm Res. 2000,17, 593-9.

 

113a21.  Eckhart L, Bach J, Ban J, Tschachler E.

 Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity.

Biochem Biophys Res Commun. 2000 ,271, 726-30.

 

113a22.  Nakahara Y, Hanajiri R.

 Hair analysis for drugs of abuse XXI. Effect of para-substituents on benzene ring of methamphetamine on drug incorporation into rat hair.

Life Sci. 2000, 66, 563-74.

 

113a23.  Lapina VA , Sheshko PM, Pankovets EA, Dontsov AE.

 Phytosorbent prepared from sunflower seed husks prevents mercuric chloride accumulation in kidney and muscle of adult rabbits.

Arch Environ Health. 2000, 55, 48-50.

 

113a24.  Ishikawa H, Mitsui Y, Yoshitomi T, Mashimo K, Aoki S, Mukuno K, Shimizu K.

 Presynaptic effects of botulinum toxin type A on the neuronally evoked response of albino and pigmented rabbit iris sphincter and dilator muscles.

Jpn J Ophthalmol. 2000, 44,106-9.

 

 

 

 

 

 

113a25.  German EJ, Wood D, Hurst MA .

 Ocular effects of antimuscarinic compounds: is clinical effect determined by binding affinity for muscarinic receptors or melanin pigment?

J Ocul Pharmacol Ther. 1999, 15, 257-69.

 

113a26.  Drozdz R, Hintermann E, Tanner H, Zumsteg U, Eberle AN.

 (D-(p-benzoylphenylalanine)13, tyrosine19)-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking.

J Pept Sci. 1999, 5, 234-42.

 

113a27.  Englaro W, Bahadoran P, Bertolotto C, Busca R, Derijard B, Livolsi A,Peyron JF, Ortonne JP, Ballotti R.

 Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

Oncogene. 1999 ,18, 1553-9.

 

113a28. Schraermeyer U, Peters S, Thumann G, Kociok N, Heimann K.

 Melanin granules of retinal pigment epithelium are connected with the lysosomal degradation pathway.

Exp Eye Res. 1999,68, 237-45.

 

113a29.  Lichter A, Mills D.

 Control of pigmentation of Ustilago hordei: the effect of pH, thiamine, and involvement of the cAMP cascade.

Fungal Genet Biol. 1998, 25, 63-74.

 

 

 

 

113a30.  Grossi GF, Durante M, Gialanella G, Pugliese M, Mosse I.

 Effects of melanin on high- and low- linear energy transfer (LET) radiationresponse of human epithelial cells.

Radiat Environ Biophys. 1998 , 37, 63-7.

 

 

113a31.  Palumbo A, Mars U, De Martino L, d'Ischia M, Napolitano A, Larsson BS, Prota G.

 Selective incorporation of the prototype melanoma seeker thiourea into nascent melanin: a chemical insight.

Melanoma Res. 1997, 7, 478-85.

 

113a32.  Hyodo-Taguchi Y, Winkler C, Kurihara Y, Schartl A, Schartl M.

 Phenotypic rescue of the albino mutation in the medakafish (Oryzias latipes) by a mouse tyrosinase transgene.

Mech Dev. 1997, 68, 27-35.

 

113a33.  Blackstaffe L, Shelley MD, Fish RG.

 Cytotoxicity of gossypol enantiomers and its quinone metabolite gossypolone in melanoma cell lines.

Melanoma Res. 1997, 7, 364-72.

 

113a34. Joseph RE Jr, Tsai WJ, Tsao LI, Su TP, Cone EJ.

 In vitro characterization of cocaine binding sites in human hair.

J Pharmacol Exp Ther. 1997, 282, 1228-41.

 

113a35.  Kordysh EA, Herishanu Y, Goldsmith JR.

 Chemical exposures and Parkinson's disease in residents of three Negev

kibbutzim.

Environ Res. 1997, 73, 162-5.

 

 

 

113a36.  Offen D, Ziv I, Panet H, Wasserman L, Stein R, Melamed E, Barzilai A.

 Dopamine-induced apoptosis is inhibited in PC12 cells expressing Bcl-2.

Cell Mol Neurobiol. 1997, 17, 289-304.

 

113a37. Rothe M, Pragst F, Thor S, Hunger J.

 Effect of pigmentation on the drug deposition in hair of grey-haired subjects.

Forensic Sci Int. 1997, 84,53-60.

 

113a38.  Fogarty RV, Tobin JM.

 Fungal melanins and their interactions with metals.

Enzyme Microb Technol. 1996, 19, 311-7. Review.

 

 

113a39.  Suzuki I, Cone RD , Im S, Nordlund J, Abdel-Malek ZA.

 Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis.

Endocrinology. 1996, 137, 1627-33.

 

113a40.  Green SJ, Wilson JF.

 The effect of hair color on the incorporation of methadone into hair in the

rat.

J Anal Toxicol. 1996, 20, 121-3.

 

113a41.  Wang Y, Casadevall A.

 Susceptibility of melanized and nonmelanized Cryptococcus neoformans to the melanin-binding compounds trifluoperazine and chloroquine.

Antimicrob Agents Chemother. 1996 , 40, 541-5.

.

113a42  Mars U, Larsson BS.

 New thioureas and related substances intended for melanoma targeting.

Pigment Cell Res. 1995, 8, 194-201.

 

113a43.  Sugumaran M, Nellaiappan K, Scott T, Amaratunga C.

 Complex formation between mushroom tyrosinase and Manduca dopachrome isomerase.

Pigment Cell Res. 1995, 8, 180-6.

 

113a44.  Haylett AK , Ross S, Truscott TG, Moore JV.

 Pharmacokinetic and therapeutic outcome in melanoma cells, of the

administration of symmetric and asymmetric cationic photosensitizers.

Cancer Lett. 1995, 88, 191-9.

 

113a45.  Hill RA, Esterowitz T, Ryan J, Liaw LH, Nelson JS, Yashiro H, Krasieva T, Berns MW.

 Photodynamic laser cyclodestruction with chloroaluminum sulfonated

phthalocyanine (CASPc) or Photofrin (PII) vs. Nd:YAG laser cyclodestruction in a pigmented rabbit model.

Lasers Surg Med. 1995,17, 166-71.

 

113a46.  Nogaj P, Buszman E, Swiatkowska L, Wilczok T.

 The effect of endo- and exogenous melanin on Zn2+ and Co2+ elimination and

distribution in mice.

Acta Biochim Pol. 1995, 42, 83-8.

 

113a47.  Trope GE, Menon IA , Liu GS, Thibodeau JR, Becker MA, Persad SD.

 Ocular timolol levels after drug withdrawal: an experimental model.

Can J Ophthalmol. 1994, 29, 217-9.

 

   

GENERAL

   

114. J.Borovansky, M.Elleder  ‘’  Melanosome degradation : fact or finction ‘’Pigment Cell Res., 16, 280-286, 2003

115. M.D’ Ischia   ‘’Melanins and melanogenesis : The Changing Landscape : A Tribute to Giuseppe Prota ‘’  Pigment Cell Res. 16, 574, 2003.

116. Y.Kawashima, A.Aoki, S.Ishii, H.Watanabe, I.Ishikawa  ‘’ Laser treatment of gingival melanin pigmentatio ‘’Intern.Congress Series, 1248, 245-248, 2003.

117. C.C.Dierickx ‘’ Lasers in pigmentary disorders  ‘’ Pigment Cell Res. 16, 590, 2003.

117a. J.D.Benigni, R.L.Minnis ‘’ The syntheses of 5,6-dihydroxyindole and some of its derivatives ‘’ J.Heterocyclic Chem., 2, 387, 1965

 

 MELANOGENESIS

  118. G.Raposo, D.Tenza,J.F.Berson, D.Harper, A.Theos, M.S.Marks  ‘’The biogenesis of melanosomes in highly pigmented cells ‘’ Pigment Cell Res. 16, 584, 2003.

119. S.Ito  ‘’ A Chemist’s View of Melanogenesis ‘’   Pigment Cell Res. 16, 230-236, 2003.

120. P.A.Riley  ‘’ Melanogenesis and melanoma  ‘’  Pigment Cell Res., 16, 548-552, 2003.

121. A,Palumbo ‘’ Melanogenesis in the ink gland of  Sepia officinalis ‘’  Pigment Cell Res. 16, 517-522, 2003.

122. V.J.Hearing ‘’ Regulation of melanogenesis in the melanosome ‘’ Pigment Cell Res., 16, 570, 2003.

123. B.Kasraee, O.Sorg, J.H.Saurat  ‘’Hydrogen peroxide in the presence of cellular antioxidants mediates the first and key step of melanogenesis :a new concept introducing melanin production as a cellular defence mechanism against oxidative stress ‘’  Pigment Cell Res., 16, 571, 2003.

   

NEUROMELANIN

  

124. K.Wakamatsu, K.Fujikawa, F.A.Zucca,L.Zucca, S.Ito  ‘’  The structure of neuromelanin as studied by chemical degradative methods ‘’    J.of Neurochemistry  86, 1015-1023, 2003.

  Substantia neuromelanin 13.doc

124a1--  Substantia nigra

 Black / brown pigmented granules are present in the human central nervous system (1-4)  The most pigmented regions are two areas: substantia nigra and the locus coeruleus. Histological studies displayed pigmentation in the substantia nigra of other mammals phylogenetically close to humans, including the shimpanzee, and more distant ones, such as horses and sheep. Histochemical studies on human substantia nigra and locus coeruleus found that the pigment had similar properties to the melanins, including  insolubility ,  bleaching by hydrogen peroxide, labelling by silver stains. The pigment was therefore named neuromelanin. Histological studies showed that neuromelanin granules were located in the neuronal perikaryon and were surrounded by a double membrane. In humans and horses, histochemical analyses indicated an association of neuromelanin granules with lipofuscin. In the substantia nigra, neuromelanin accumulates during aging  and is found after the first 2 to 3 years of life. The  substantia nigra neurones are more vulnerable than the non-pigmented ones. However, important questions remain regarding the possible role of neuromelanin in the substantia nigra, both under physiological conditions and in the pathogenesis of Parkinson’s disease. Neuromelanins like others melanins (BCM) show electrical activity.The presence in the brain of electrical material may be interesting.

Dopamine  may be a precursor of neuromelanin  but like other melanins the polymer has more oxygen than the precursor.

 Dopamine ( C8H11NO2 , mol.weight 153,18 ;  C% 62.7,  H% 7.2,    N%9.1  ) forms in alkaline medium, by oxidation,  black / brown material..Dopamine is present in bananas being responsible for the blackning of the fruit.Dopamine partecipate to  the biogenesis of some  alkaloids .

Initially, the name neuromelanin was chosen because of its similarity in appearance to skin melanin. However, the electron paramagnetic resonance (EPR) and metal analysis studies indicate that chemically neuromelanin   has a stable free radical structure. Furthermore neuromelanin and synthetic melanin are amorphous semiconductors.

The ability of neuromelanin to bind several inorganic and organic compounds, to chelate metal ions , to store liquids and gases, to show sound and electrical conductivity,  are difficult to understand from a chemical point of view but not from the nanoscientists.

Degradation analyses using potassium permanganate and hydriodic acid hydrolysis showed that neuromelanin has properties of both pheomelanin and eumelanin. Elemental analyses of neuromelanin revealed a high sulphur content (2.5–2.8%), with a molar C/H ratio lower than that of synthetic melanins, thus indicating the presence of aliphatic groups and benzothiazine rings.

Infrared spectroscopy of neuromelanin ( and other melanins BCM ) revealed the presence of aliphatic groups and a low intensity aromatic component, whereas in synthetic melanins the aliphatic groups were absent. Chemical degradation studies showed that neuromelanin  is a mixture of melanin (BCM)  and pheomelanin  ( see Link   14 ) .

Neuromelanin  shows a peptide component of about 15%. The amino acids could be derived from a direct reaction between the melanic polymer and proteins, or dopamine molecules bound to cysteinic residues of polypeptidic chains. Indeed, the precursor of neuromelanin synthesis has been suggested to be cysteinyl-dopamine, although a study using hydriodic acid hydrolysis failed to identify the corresponding degradation products. Moreover, nuclear magnetic resonance spectroscopy indicates that the presence of both aliphatic and aromatic hydrogens, and the ratio of aliphatic to aromatic hydrogens is again higher in neuromelanin than in synthetic melanins, suggesting that dopamine cannot be the only precursor in neuromelanin synthesis.

x Ray difraction studies have shown that neuromelanin has a multilayer (graphite-like) three dimensional structure similar to synthetic and naturally occurring melanins. The three dimensional structure is derived from planar overlapped sheets consisting of cyclic molecules of indolebenzothiazine rings.

 

However,these sheets are stacked much higher in neuromelanin than in any other synthetic and naturally occurring melanins.

 

The process of neuromelanin formation is difficult to understand.  It has long been debated whether the synthesis of neuromelanin is enzymatically mediated or whether it is a pure autooxidation process of dopamine derivatives. For eumelanin synthesis, the enzyme tyrosinase   catalyses the conversion of tyrosine to L-dopa and then to dopa-quinone. Some authors proposed that tyrosinase could also be involved in neuromelanin biosynthesis because tyrosinase mRNA and promoter activity have been detected in the substantia nigra.

Moreover, albinos who lack tyrosinase display normally pigmented substantia nigra. Alternative enzymatic actions have been suggested, including tyrosine hydroxylase mediated oxidation of dopamine. In another study, peroxidase catalysed the oxidation of tyrosine to dopa and then dopamine, and further oxidisation to the respective quinones that are possible precursors of neuromelanin.

Alternatively, neuromelanin could derive from non-enzymatic oxidation. The autooxidation  of catechols to quinones with the addition of a thiol has been demonstrated in the brain.

A dopamine-melanin can be synthesised by the autooxidation of dopamine, although there are several structural differences between synthetic melanins and the natural one isolated from the substantia nigra. Recently, neuromelanin synthesis was induced in rat substantia nigra neurones and PC cell cultures by exposure to L-dopa. The pigment produced in this model contains a stable free radical; in addition, both light and electron microscopy have shown that the pigment synthesised in these cells appears to be identical to human neuromelanin, and the granules are surroundedby a double membrane, similar to the naturally occurring neuromelanin of the substantia nigra. In those experiments, treatment with the iron chelator desferrioxamine inhibited neuromelanin synthesis stimulated by L-dopa; therefore, it seems that iron ( or an iron catalyst ) is involved in neuromelanin formation. In this model, neuromelanin synthesis was shown to be driven by an excess of cytosolic catecholamines not accumulated in synaptic vesicles.

The herbicide paraquat has a molecular structure similar to that of MPTP, and has been proposed as a Parkinson’s disease inducing agent. The pesticide is accumulated in neuromelanin containing nerve cells, where it appeared that the neuromelanin adsorbed intraneuronal paraquat, protecting the neurones from consequent damage.

Neuromelanin like others BCM can also accumulate chlorpromazine, haloperidol, and imipramine, thereby contributing to the control of the intraneuronal concentration of these molecules.

 Because higher intraneuronal concentrations of dopaminergic drugs might be toxic to substantia nigra neurones, neuromelanin can influence this toxicity. The association of neuromelanin with lipids has been described in several studies.

Although previous studies proposed that lipids were part of the neuromelanin molecule, recent work has shown that neuromelanin contains about 20% adsorbed lipids. Cholesterol is a minor component in this lipid mixture, with the major component being a new class of polyunsaturated lipid with a high molecular weight, low volatility, and low oxygen content.

High concentrations of iron and other  metals are present in several brain nuclei. Neuromelanin from the substantia nigra can interact with many heavy metal ions such as zinc, copper, manganese, chromium, cobalt, mercury, lead, and cadmium; in addition, it binds iron particularly EPR studies showed that in the substantia nigra the ferric iron is bound to     neuromelanin as a high spin complex with an octahedral configuration. Mössbauer spectroscopy demonstrates that ferric iron is       chelated by the neuromelanin polymer and that the iron sites are arranged in a ferritin-like iron-oxy-hydroxide cluster form. The cluster conductivity is unknown.

X rays absorption fine structure spectroscopy and infrared spectroscopy studies confirmed thatiron in neuromelanin was bound by oxygen derived phenolic groups in an octahedral configuration. In substantia nigra tissue, neuromelanin is only about 50% saturated withFe(III), therefore maintaining an important residual chelating capability, which can protect against iron toxicity.Values of iron complex conductivity are unknown.

Neuromelanin can sequester redox active iron ions, reducing the formation of free hydroxyl radicals. Thus, in normal subject s, neuromelanin may play a protective role b    inactivating the iron ions that induce oxidative   stress. The ability of neuromelanin to chelate other redox active metals such as copper, manganese, chromium, and toxic metals including cadmium, mercury, and lead  strengthens the hypothesis that neuromelanin may have a high capacity storage trapping system for metal ions which  may prevent neuronal damage.

Electrical conductivity of the different chelates has not determined until now. Neuromelanin accumulates normally with age in human substantia nigra neurones. A neuronal pigment has also been observed in the ubstantia nigra of adult rats and dogs, and its concentration seems to depend upon age. In very old (23 months) rats, but not in younger animals, neuromelanin granules were detected by electron microscopy; similar results were observed in aged dogs. Neuromelanin granules were also detected in catecholaminergic cerebellar cells of monkeys  and their presence correlated with age. In human substantia nigra, the first small, brown neuromelanin granules were clearly discernable at approxim tely 3 to 5 years of age. The neuromelanin content of neurones is highest in individuals in th ir 60s, after which it decreases; this phenomenon may reflect the neuronal loss observed in these anatomical structures during aging. However, there is no significant loss of catecholaminergic neurones in the substantia nigra of normal subjects until very old ages.

In patients with Parkinson’s disease, neuromelanin values were 1.2–1.5 mg/g of substantia nigra , which is less than 50% of that seen in age matched controls . The absolute number of pigmented neurones in the substantia nigra of normal subjects may be dependent upon ethnicity—an Indian population was found to have fewer pigmented neurones than an agematched Western population.. Because the neuromelanin concentration in substantia nigra neurones increases, and the number of pigmented neurones appears to be constant over the life span, it seems that neuromelanin accumulates only in a subpopulation of nigral neurones, whereas other dopaminergic neurones remain nonpigmented. The observed decrease in the neuromelanin concentration occurring in the substantia nigra of patients with Parkinson’s disease confirms the loss of pigmented neurones occurring in the substantia nigra of these patients, as has been reported in neuropathological studies. Other studies indicate that neuromelanin values decrease in the surviving neurones of the substantia nigra during Parkinson’s disease. This could be the result of reduced neuromelanin synthesis, neuromelanin degradation, or higher vulnerability of the pigmented neurones.

Although in idiopathic Parkinson’s disease the neurones are depleted in both the substantia nigra and locus coeruleus, in MPTP intoxicated subjects, locus coeruleus neurones are spared. Such a different neuronal vulnerability might eventually be explained by structural differences in the neuromelanin of the substantia nigra and locus coeruleus.

Although neuromelanin may play a cytoprotective role by sequestering redox active metals, toxic metals, and organic toxic compounds, neuromelanin might also become a source of free radicals by reaction with hydrogen peroxide. When free neuronal iron increases to the point where neuromelanin becomes saturated and it starts to catalyse the production of free radicals, neuromelanin would become cytotoxic with an increasing of the conductivity.

 Moreover, because hydrogen peroxide can degrade neuromelanin, the pigmented neurones could loose this putatively protective agent. The consequence may be a release of iron and other cytotoxic metals or compounds from neuromelanin that could accelerate neuronal death.

 

  To sum up  :  the precursor of neuromelanin is unknown

 

References :

 1) .  Schrerer HJ. Melanin pigmentation of the substantia nigra

in primates. J Comp Neurol  71, 91-97, 1939.

 2) .  R.A.Nicolaus Melanins pag 9, 67, 103,  Hermann, Paris 1968

 3) .  G.Prota  Melanins and melanogenesis  pag 119-133, AP, San Diego 1992

 4) . L.Zecca, D.Tampellini, M.Gerlach, P.Riederer, R.G.Fariello, D.Sulzer,   Substantia nigra neuromelanin : structure , synthesis, and molecular behaviour J.Clin. Pathol: Mol.Pathol. 54, 414-418, 2001 ;  L.Zecca, F.A.Zucca, P.Costi, D.Tampellin, A.Gatti, M.Gerlach, P.Riederer, R.G.Fariello, S.Ito, M.Gallorini, D.Sulzer  The neuromelanin of human Substantia nigra : structure, synthesis, and molecular behaviour    J.Neural. Transm. Suppl., 65, 145-155, 2003

   

124a2. Melanins:Opening Doors into Biological Conductors

Rodolfo A. Nicolaus*, Adele Bolognese, Antonio Lavecchia§, Orazio Mazzoni§, Andrea Cavalli, Barbara Nicolaus & Ettore Novellino§

 

*Accademia Pontaniana, via Mezzocannone 8, I-80134 Napoli, Italy

Dipartimento di Chimica Organica e Biochimica, via Cynthia Monte Sant’Angelo Università di Napoli “Federico II”, I-80126, Italy.

§Dipartimento di Chimica Farmaceutica e Tossicologica, via D. Montesano 49, Università di Napoli “Federico II”, I-80131, Italy.

Scuola Internazionale Superiore di Studi Avanzati (SISSA/ISAS), via Beirut 2-4, I-34014 Trieste, Italy

Laboratorio per le molecole di interesse biologico. CNR, Arco Felice , Napoli.

 

          Melanins, the organic black pigments, present on earth as in stellar interspaces are radical-polarone macromolecules, characterized by electronic lacunae and by positive charges located on an extensively conjugated carbonium skeleton.

          Here we report a structural study of the most widespread melanin, sepiomelanin, and of its synthetic model, 5,6-dihydroxyindole melanin (DHI-melanin). The natural black and the synthetic pigment are described as filaments which are mainly constituted by repetitive units of a gem-diol, the 5-dihydroxyindol-6-one (DHIO), bonded at positions 4 and 7.

          The linear strands in two low-energy conformational states, referred as helix and alternating-sheet, can arrange themselves to form strata and cages, accounting for the capability of melanin to absorb gas and ions and to form metal chelates. A three-dimensional model, constituted by three DHIO units, exhibits a small HOMO-LUMO gap, supporting evidence which indicates melanin as a highly conductive organic semiconductor.

          Structural, conformational and electrical properties can explain the different roles of melanins in different biological districts, from the epithelial tissues to the dopaminergic neurons of substantia nigra.

 

Structure of melanin and of acetylene black.

Side and front view of helix conformation

 

 

Side and front view of alternating-sheet conformation

 

 

 

 

McGinness, J. E., Corry, P. M. & Proctor, P. H. Science 183, 853-855 (1974).

Filatovs, J. E., Corry, P. M. & McGinness, J. E. Biopolymers 15, 2309-2312 (1976).

   

124a3.  Neuromelanin of the substantia nigra: a neuronal black hole with protective and toxic characteristics.

Zecca L, Zucca FA, Wilms H, Sulzer D.


Trends Neurosci. 26, 578-580, 2003

Neuromelanin accumulates in dopaminergic neurons during normal aging, and in Parkinson's disease, neurons with this pigment are those that selectively degenerate. Intraneuronal neuromelanin could play a protective role during its synthesis by preventing the toxic accumulation of cytosolic catechol derivatives and, in addition, by its ability to scavenge reactive metals, pesticides and other toxins to form stable adducts. However, dying neurons in Parkinson's disease that release neuromelanin might induce a vicious cycle of chronic neuroinflammation and neuronal loss.

 124a4. The neuromelanin of human substantia nigra: structure, synthesis and molecular behaviour.

Zecca L, Zucca FA, Costi P, Tampellini D, Gatti A, Gerlach M, Riederer P, Fariello RG, Ito S, Gallorini M, Sulzer D.

 

J.Neural. Transm. Suppl., 65, 145-155, 2003

 luigi.zecca@itb.cnr.it

The pigmented neurons of the substantia nigra (SN) are typically lost in Parkinson's disease: however the possible
relationship between neuronal vulnerability and the presence of neuromelanin (NM) has not been elucidated. Early histological studies revealed the presence of increasing amounts of NM in the SN with aging in higher mammals, showed that NM granules are surrounded by membrane, and comparatively evaluated the pigmentation of SN in different animal species. Histochemical studies showed the association of NM with lipofuscins. However, systematic investigations of NM structure, synthesis and molecular interactions have been undertaken only during the last decade. In these latter studies, NM was identified as a genuine melanin with a strong chelating ability for iron and affinity for compounds such as lipids, pesticides, and MPP+. The affinity of NM for a variety of inorganic and organic toxins is consistent with a postulated protective function for NM. Moreover, the neuronal accumulation of NM during aging, and the link between its synthesis and high cytosolic concentration of catechols suggests a protective role. However, its putative neuroprotective effects could be quenched in conditions of toxin overload.


124a5.  THE EFFECT OF HUMAN AND SYNTHETIC NEUROMELANIN ON NEURONAL AND GLIAL CELL LINES

 *F.M. Griffiths, *K.L. Double and **D. Rowe

**Prince of Wales Medical Research Institute, Randwick , 2031 and the **Department of

Neurology, Royal North Shore Hospital , St Leonards. 2065, Sydney , Australia

 The dopaminergic neurons of the substantia nigra contain a dark-coloured pigment called neuromelanin (NM). The function of NM is unknown but, like other melanins in the body, may protect the cell against oxidative damage. To date, most studies on NM have utilised a synthetic dopamine melanin (DAM), rather than the native pigment. The aim of the present study was to determine the relative effects of NM and DAM upon human-derived neuroblastoma (SK-N-SH) and glioblastoma (U373) cell lines in the presence and absence of an oxidative stimulus. Cellswere incubated for 24 hours in the presence of isolated human NM or DAM, with or without

Fenton reagent. Cell damage was assessed by measurement of lactate dehydrogenase (LDH) activity and mitochondrial function. LDH activity in glial cells was unaffected by all treatments.In neuronal cells, LDH activity was increased to 165% of basal levels in the presence of DAM

and decreased to 80% of basal levels in the presence of NM. Fenton reagent significantly increased LDH activity to 170% of basal levels. The addition of DAM, but not NM, to Fentonreagent was additive (333% of basal LDH). Mitochondrial redox status and function was

unaffected by any treatment over a period of 24 hours, in both cell lines. Our results suggest that DAM is a poor model of NM. DAM, but not NM, induces cellular damage in neuronal cells.

 Further, the effect of an oxidative stimulus is intensified by DAM but not by NM.

 Substantia 7.doc

124a6.  Ostergren A, Annas A, Skog K, Lindquist NG, Brittebo EB.

 Long-term retention of neurotoxic beta-carbolines in brain neuromelanin.

J Neural Transm. 2004 Feb 12;111(2):141-157.


124a7.  Marco FD, Foppoli C, Coccia R, Blarzino C, Perluigi M, Cini C, Marcante ML. 

 Ectopic deposition of melanin pigments as detoxifying mechanism: a paradigm for

basal nuclei pigmentation.

Biochem Biophys Res Commun. 2004 Feb 6;314(2):631-7.

 124a8.  Double KL, Halliday GM, Henderson J, Griffiths FM, Heinemann T, Riederer P,

Gerlach M. 

 The dopamine receptor agonist lisuride attenuates iron-mediated dopaminergic

neurodegeneration.

Exp Neurol. 2003 Nov;184(1):530-5. 

124a9.  Zecca L, Zucca FA, Wilms H, Sulzer D. 

 Neuromelanin of the substantia nigra: a neuronal black hole with protective and

toxic characteristics.

Trends Neurosci. 2003 Nov;26(11):578-80. Review. 

124a10.  Connor JR, Boyer PJ, Menzies SL, Dellinger B, Allen RP, Ondo WG, Earley CJ. 

 Neuropathological examination suggests impaired brain iron acquisition in

restless legs syndrome.

Neurology. 2003 Aug 12;61(3):304-9. 

124a11.  Faucheux BA, Martin ME, Beaumont C, Hauw JJ, Agid Y, Hirsch EC. 

 Neuromelanin associated redox-active iron is increased in the substantia nigra

of patients with Parkinson's disease.
J Neurochem. 2003 Sep;86(5):1142-8. 


124a12.  Double KL, Gerlach M, Schunemann V, Trautwein AX, Zecca L, Gallorini M,

Youdim MB, Riederer P, Ben-Shachar D. 

Iron-binding characteristics of neuromelanin of the human substantia nigra.

Biochem Pharmacol. 2003 Aug 1;66(3):489-94. 
 
124a13.  Wakamatsu K, Fujikawa K, Zucca FA, Zecca L, Ito S. 

 The structure of neuromelanin as studied by chemical degradative methods.

J Neurochem. 2003 Aug;86(4):1015-23. 

124a14.  Fahn S. 

 Description of Parkinson's disease as a clinical syndrome.

Ann N Y Acad Sci. 2003 Jun;991:1-14. Review. 


124a15.  Wilczok T, Stepien K, Dzierzega-Lecznar A, Zajdel A, Wilczok A. 

 Model neuromelanins as antioxidative agents during lipid peroxidation.

Neurotox Res. 1999 Dec;1(2):141-7. 

124a16.  Sulzer D, Zecca L. 

 Intraneuronal dopamine-quinone synthesis: a review.

Neurotox Res. 2000 Feb;1(3):181-95. 

124a17.  Solano F, Hearing VJ, Garcia-Borron JC. 

 Neurotoxicity due to o-quinones: neuromelanin formation and possible mechanismsfor o-quinone detoxification.

Neurotox Res. 2000 Feb;1(3):153-69. 

PMID: 12835099 [PubMed]

124a18.  Gerlach M, Double KL, Ben-Shachar D, Zecca L, Youdim MB, Riederer P. 

 Neuromelanin and its interaction with iron as a potential risk factor for

dopaminergic neurodegeneration underlying Parkinson's disease.

Neurotox Res. 2003;5(1-2):35-44. Review. 

124a19.  Smythies J, De Iuliis A, Zanatta L, Galzigna L. 
 The biochemical basis of Parkinson's disease: the role of catecholamine

o-quinones: a review-discussion.

Neurotox Res. 2002 Feb;4(1):77-81. 

PMID: 12826496 [PubMed - in process]

124a20  Chan AC, Ho LC, Yip WW, Cheung FC. 

 Pigmented ependymoma with lipofuscin and neuromelanin production.

Arch Pathol Lab Med. 2003 Jul;127(7):872-5. 

124a21.  Jellinger KA. 

 Rett Syndrome -- an update.

J Neural Transm. 2003 Jun;110(6):681-701. Review. 


124a22.  Iihara K, Yamaguchi K, Fujioka Y, Uno S. 

 Pigmented neuroendocrine tumor of the lung, showing neuromelanin.

Pathol Int. 2002 Nov;52(11):734-9. 

124a23.  Reyes MG, Faraldi F, Rydman R, Wang CC. 

 Decreased nigral neuromelanin in Alzheimer's disease.
Neurol Res. 2003 Mar;25(2):179-82. 


 124a24. Becker G. 

 [Methods for the early diagnosis of Parkinson's disease]

Nervenarzt. 2003 Mar;74 Suppl 1:S7-11. German. 


124a25.  Fasano M, Giraudo S, Coha S, Bergamasco B, Lopiano L. 

 Residual substantia nigra neuromelanin in Parkinson's disease is cross-linked

to alpha-synuclein.

Neurochem Int. 2003 Jun;42(7):603-6. 


124a26.  Usunoff KG, Itzev DE, Ovtscharoff WA, Marani E. 
 Neuromelanin in the human brain: a review and atlas of pigmented cells in the
substantia nigra.

Arch Physiol Biochem. 2002 Oct;110(4):257-369. Review. 


124a27.  Double KL, Ben-Shachar D, Youdim MB, Zecca L, Riederer P, Gerlach M. 

 Influence of neuromelanin on oxidative pathways within the human substantia
nigra.

Neurotoxicol Teratol. 2002 Sep-Oct;24(5):621-8. Review. 

124a28.  Collins MA, Neafsey EJ. 

 Potential neurotoxic "agents provocateurs" in Parkinson's disease.

Neurotoxicol Teratol. 2002 Sep-Oct;24(5):571-7. Review. 

124a29.  Kameyama K, Takami H. 

 Pigmented Granules in Functional Black Adenoma of the Adrenal Gland: A

Histochemical and Ultrastructural Study.

Endocr Pathol. 1999 Winter;10(4):353-357. 

124a30.  Zecca L, Tampellini D, Gatti A, Crippa R, Eisner M, Sulzer D, Ito S,

Fariello R, Gallorini M. 

 The neuromelanin of human substantia nigra and its interaction with metals.
J Neural Transm. 2002 May;109(5-6):663-72. Review. 

124a31.  Stepien K, Wilczok A, Zajdel A, Dzierzega-Lecznar A, Wilczok T. 

 Peroxynitrite mediated linoleic acid oxidation and tyrosine nitration in the

presence of synthetic neuromelanins.

Acta Biochim Pol. 2000;47(4):931-40. 

124a32.  Bolzoni F, Giraudo S, Lopiano L, Bergamasco B, Fasano M, Crippa PR. 

 Magnetic investigations of human mesencephalic neuromelanin.

Biochim Biophys Acta. 2002 Mar 16;1586(2):210-8. 

124a33.  Zecca L, Fariello R, Riederer P, Sulzer D, Gatti A, Tampellini D. 

 The absolute concentration of nigral neuromelanin, assayed by a new sensitive

method, increases throughout the life and is dramatically decreased in

Parkinson's disease.

FEBS Lett. 2002 Jan 16;510(3):216-20. 

124a34.  Drukarch B, van Muiswinkel FL. 

 Neuroprotection for Parkinson's disease: a new approach for a new millennium.

Expert Opin Investig Drugs. 2001 Oct;10(10):1855-68. Review. 


124a35.  Schneider JA, Bienias JL, Gilley DW, Kvarnberg DE, Mufson EJ, Bennett DA. 

 Improved detection of substantia nigra pathology in Alzheimer's disease.
J Histochem Cytochem. 2002 Jan;50(1):99-106. 

124a36.  Barzilai A, Melamed E, Shirvan A. 

 Is there a rationale for neuroprotection against dopamine toxicity in

Parkinson's disease?

Cell Mol Neurobiol. 2001 Jun;21(3):215-35. Review. 

124a37.  Elleder M, Borovansky J. 

 Autofluorescence of melanins induced by ultraviolet radiation and near

ultraviolet light. A histochemical and biochemical study.

Histochem J. 2001 May;33(5):273-81. 

124a38.  Yoshida S, Ektessabi A, Fujisawa 

 XANES spectroscopy of a single neuron from a patient with Parkinson's disease.

J Synchrotron Radiat. 2001 Mar 1;8(Pt 2):998-1000. 

 
124a39.  Chauhan NB, Siegel GJ, Lee JM. 

 Depletion of glial cell line-derived neurotrophic factor in substantia nigra

neurons of Parkinson's disease brain.

J Chem Neuroanat. 2001 Jun;21(4):277-88. 

124a40.  Zecca L, Gallorini M, Schunemann V, Trautwein AX, Gerlach M, Riederer P,

Vezzoni P, Tampellini D. 

 Iron, neuromelanin and ferritin content in the substantia nigra of normal

subjects at different ages: consequences for iron storage and neurodegenerative

processes.
J Neurochem. 2001 Mar;76(6):1766-73. 
 
124a41.  Smythies J. 

 Redox aspects of signaling by catecholamines and their metabolites.

Antioxid Redox Signal. 2000 Fall;2(3):575-83. Review. 


124a42.  Nappi AJ, Vass E. 

 The effects of nitric oxide on the oxidations of l-dopa and dopamine mediated

by tyrosinase and peroxidase.

J Biol Chem. 2001 Apr 6;276(14):11214-22. Epub 2001 Jan 02. 

124a43.  Double KL, Zecca L, Costi P, Mauer M, Griesinger C, Ito S, Ben-Shachar D,

Bringmann G, Fariello RG, Riederer P, Gerlach M. 

 Structural characteristics of human substantia nigra neuromelanin and synthetic
dopamine melanins.
J Neurochem. 2000 Dec;75(6):2583-9. 

 
124a44.  Sulzer D, Bogulavsky J, Larsen KE, Behr G, Karatekin E, Kleinman MH, Turro

N, Krantz D, Edwards RH, Greene LA, Zecca L. 

 Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not

accumulated by synaptic vesicles.

Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11869-74. 

124a45.  Stepien K, Zajdel A, Wilczok A, Wilczok T, Grzelak A, Mateja A, Soszynski

M, Bartosz G. 

 Dopamine-melanin protects against tyrosine nitration, tryptophan oxidation and

Ca(2+)-ATPase inactivation induced by peroxynitrite.

Biochim Biophys Acta. 2000 Oct 18;1523(2-3):189-95. 


124a46.  Hearing VJ. 

 The melanosome: the perfect model for cellular responses to the environment.

Pigment Cell Res. 2000;13 Suppl 8:23-34. 
 
124a47.  Aime S, Bergamasco B, Casu M, Digilio G, Fasano M, Giraudo S, Lopiano L. 

 Isolation and 13C-NMR characterization of an insoluble proteinaceous fraction

from substantia nigra of patients with Parkinson's disease.

Mov Disord. 2000 Sep;15(5):977-81. 

124a48.  Linert W, Jameson GN. 

 Redox reactions of neurotransmitters possibly involved in the progression of

Parkinson's Disease.

J Inorg Biochem. 2000 Apr;79(1-4):319-26. Review. 

124a49.  Braak H, Rub U, Sandmann-Keil D, Gai WP, de Vos RA, Jansen Steur EN, Arai

K, Braak E. 

 Parkinson's disease: affection of brain stem nuclei controlling premotor and

motor neurons of the somatomotor system.

Acta Neuropathol (Berl). 2000 May;99(5):489-95. 

124a50.  Fukuda T, Igarashi T, Hiraki H, Yamaki T, Baba K, Suzuki T. 

 Abnormal pigmentation of schwannoma attributed to excess production of

neuromelanin-like pigment.

Pathol Int. 2000 Mar;50(3):230-7. 

124a51.  Zecca L, Costi P, Mecacci C, Ito S, Terreni M, Sonnino S. 

 Interaction of human substantia nigra neuromelanin with lipids and peptides.

J Neurochem. 2000 Apr;74(4):1758-65. 


124a52.  Lopiano L, Chiesa M, Digilio G, Giraudo S, Bergamasco B, Torre E, Fasano M.

Q-band EPR investigations of neuromelanin in control and Parkinson's disease

patients.

Biochim Biophys Acta. 2000 Mar 17;1500(3):306-12. 


124a53.  Zhu MY, Klimek V, Dilley GE, Haycock JW, Stockmeier C, Overholser JC,

Meltzer HY, Ordway A.
 
 Elevated levels of tyrosine hydroxylase in the locus coeruleus in major

depression.

Biol Psychiatry. 1999 Nov 1;46(9):1275-86. 

124a54.  Bridelli MG, Tampellini D, Zecca L. 

 The structure of neuromelanin and its iron binding site studied by infrared
spectroscopy.

FEBS Lett. 1999 Aug 20;457(1):18-22. 


124a55.  Mars U, Larsson BS. 

 Pheomelanin as a binding site for drugs and chemicals.

Pigment Cell Res. 1999 Aug;12(4):266-74. 

124a56.  Ma SY, Ciliax BJ, Stebbins G, Jaffar S, Joyce JN, Cochran EJ, Kordower JH,

Mash DC, Levey AI, Mufson EJ. 

 Dopamine transporter-immunoreactive neurons decrease with age in the human

substantia nigra.

J Comp Neurol. 1999 Jun 21;409(1):25-37. 


124a57.  Jellinger KA. 

 The role of iron in neurodegeneration: prospects for pharmacotherapy of

Parkinson's disease.

Drugs Aging. 1999 Feb;14(2):115-40. Review. 


124a58.  Offen D, Gorodin S, Melamed E, Hanania J, Malik Z. 

 Dopamine-melanin is actively phagocytized by PC12 cells and cerebellar granular

cells: possible implications for the etiology of Parkinson's disease.

Neurosci Lett. 1999 Jan 29;260(2):101-4. 


124a59.  Fyffe WE, Kronz JD, Edmonds PA, Donndelinger TM. 

 Effect of high-level oxygen exposure on the peroxidase activity and the

neuromelanin-like pigment content of the nerve net in the earthworm, Lumbricus

terrestris.

Cell Tissue Res. 1999 Feb;295(2):349-54. 


124a60.  Kropf AJ, Bunker BA, Eisner M, Moss SC, Zecca L, Stroppolo A, Crippa PR. 

 X-ray absorption fine-structure spectroscopy studies of Fe sites in natural

human neuromelanin and synthetic analogues.

Biophys J. 1998 Dec;75(6):3135-42. 


124a61.  Li H, Shen XM, Dryhurst G. 

 Brain mitochondria catalyze the oxidation of

7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxyli c acid

(DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge

glutathione: potential relevance to the pathogenesis of Parkinson's disease.

J Neurochem. 1998 Nov;71(5):2049-62. 


124a62.  Ikemoto K, Nagatsu I, Ito S, King RA, Nishimura A, Nagatsu T. 

 Does tyrosinase exist in neuromelanin-pigmented neurons in the human substantia

nigra?

Neurosci Lett. 1998 Sep 11;253(3):198-200. 

 
124a63.  Nishio T, Furukawa S, Akiguchi I, Sunohara N. 

 Medial nigral dopamine neurons have rich neurotrophin support in humans.

Neuroreport. 1998 Aug 24;9(12):2847-51. 

124a64.   Cheng FC, Kuo JS, Chia LG, Dryhurst G. 
 Elevated 5-S-cysteinyldopamine/homovanillic acid ratio and reduced homovanillic

acid in cerebrospinal fluid: possible markers for and potential insights into

the pathoetiology of Parkinson's disease.

J Neural Transm. 1996;103(4):433-46. 

124a65.  Smythies J, Galzigna L. 

 The oxidative metabolism of catecholamines in the brain: a review.

Biochim Biophys Acta. 1998 Apr 10;1380(2):159-62. Review. 

124a66.  Ito S, Wakamatsu K. 

 Chemical degradation of melanins: application to identification of

dopamine-melanin.

Pigment Cell Res. 1998 Apr;11(2):120-6. 

124a67.  Tief K, Schmidt A, Beermann F. 

 New evidence for presence of tyrosinase in substantia nigra, forebrain and

midbrain.

Brain Res Mol Brain Res. 1998 Jan;53(1-2):307-10. 

124a68.  Stepien K, Zajdel A, Swierczek G, Wilczok A, Wilczok T. 

 Reduction of 13-hydroperoxy-9,11-octadecadienoic acid by dopamine-melanin.

Biochem Biophys Res Commun. 1998 Mar 27;244(3):781-4. 

124a69.  Lack EE, Kim H, Reed K. 

 Pigmented ("black") extraadrenal paraganglioma.

Am J Surg Pathol. 1998 Feb;22(2):265-9. 

 
MALDI 

124a70. The melanosome: threshold temperature for  explosive vaporization and internal absorption coefficient during pulsed laser irradiation.

S.L.Jacques , D.J. McAuliffe

 

Photochem.Photobiol., 53, 769-775, 1991

 
The explosive vaporization of melanosomes in situ in skin during pulsed laser irradiation (pulse duration less than 1 microsecond) is observed as a visible whitening of the superficial epidermal layer due to stratum corneum disruption. In this study, the ruby laser (694 nm) was used to determine the threshold radiant exposure, H0 (J/cm2), required to elicit whitening for in vitro black (Negroid) human skin samples which were pre-equilibrated at an initial temperature, Ti, of 0, 20, or 50 degrees C. A plot of H0 vs Ti yields a straight line whose x-intercept indicates the threshold temperature of explosive vaporization to be 112 +/- 7 degrees C (SD, N = 3). The slope, delta H0/delta Ti, specifies the internal absorption coefficient, mua, within the melanosome: mua = -rho C/(slope(1 + 7.1 Rd)), where rho C is the product of density and specific heat, and Rd is the total diffuse reflectance from the skin. A summary of the absorption spectrum (mua) for the melanosome interior (351-1064 nm) is presented based on H0 data from this study and the literature. The in vivo absorption spectrum (380-820 nm) for human epidermal melanin was measured by an optical fiber spectrophotometer and is compared with the melanosome spectrum.

In vivo BCM LASER degradation

 

#######################################################################################################################

     125.  MALDI  Selected papers    

 

Fragmentation occurs with heterocycle compounds and with melanins nevertheless MALDI is the most powerful tool for chemical structural studies of melanins and  particles.Samples of pure granules are required.

 

 

 

125bis. MALDI : generalità

 Le molecole di un campione di polimero non hanno tutte lo stesso peso molecolare. Si ha una distribuzione dei pesi. C'è un picco del peso molecolare medio, che viene denominato peso molecolare medio numerico o Mn. Naturalmente ci saranno sempre catene polimeriche con pesi maggiori e minori del peso molecolare medio. Se tracciamo un grafico dove abbiamo il peso molecolare sull'asse-x ed il numero delle catene che hanno un peso molecolare specifico sull'asse-y, otteniamo questo grafico:

 

 Un metodo per ottenere questo tipo di grafico è quello di utilizzare la cromatografia di esclusione sterica o SEC. Ma la SEC non misura effettivamente il peso molecolare, misura il volume idrodinamico, ossia la dimensione della catena polimerica raggomitolata in una soluzione. Questo ci da un misura relativa del peso molecolare , in quanto ovviamente più è elevato il peso molecolare più grande sarà il volume idrodinamico. A volte però è necessario sapere l'esatta distribuzione del peso molecolare. Per questo è stato inventato un processo denominato spettroscopia MALDI, ovvero spettroscopia di massa mediante desorbimento laser e ionizzazione assistita dalla matrice. Viene anche denominata solamente MALDI. Prendiamo il polimero e lo sciogliamo in acqua .    Si usa di norma una miscela 70:30 di acqua e acetonitrile . Altri solventi in cui il polimero si sciolga possono essere usati .

Si aggiunge inoltre un  composto come l'acido trans-cinnamico o  diidrossibenzoico un composto cioè che deve assorbire UV. Normalmente usiamo una quantità di assorbitore UV maggiore di 104   rispetto al nostro polimero.  Una volta miscelato il tutto il campione viene posto in una camera a tenuta d'aria, sull'estremità della sonda campione Con una pompa a vuoto risucchiamo tutta l'aria presente nella camera, o almeno quasi tutta. Mentre facciamo questo il solvente evapora e rimane uno strato del nostro composto UV assorbitore, contenente una piccola quantità di polimero. Noi diciamo che il polimero ora è disperso in una matrice del composto UV assorbirtore. Questo è il processo denominato spettroscopia di massa "MALDI".

In genere il campione viene irradiato da un laser a ultravioletti a 330-360 nm che assorbe tutta l'energia attraverso le molecole del polimero. Il materiale matrice inoltre reagisce con i polimeri in modo da trasformare i polimeri in ioni con carica elettrica come non si sa. Assorbendo tutta quella energia alcune molecole polimeriche  evaporano. Di solito le molecole polimeriche sono troppo grandi e pesanti per evaporare, ma a queste temperature così elevate con pressioni così basse, possono farlo. Ecco l'origine del termine desorbimento.  Alla fine della camera dove avviene l'evaporazione delle molecole del nostro polimero abbiamo messo due elettrodi, un catodo positivo e un anodo negativo. A seconda del tipo di polimero e al tipo di matrice utilizzata i polimeri possono diventare anioni o cationi. Per questa spiegazione consideriamo i polimeri che formano cationi positivi. Quando vaporizziamo il nostro polimero, lo vaporizziamo proprio tra i due elettrodi. Quando il nostro polimero forma i cationi, posizioniamo il catodo positivo proprio dietro il campione e l'anodo negativo davanti al campione. Naturalmente i polimeri con carica positiva si dirigeranno verso l'anodo, attratti dalla sua carica negativa. Se abbiamo agito correttamente possiamo utilizzare questa accelerazione per spingere le molecole di polimero verso il rivelatore nel misuratore posto nella parte finale della camera. La maggior parte delle volte, si ha una sola carica positiva su ogni molecola di polimero. Questo significa che la stessa forza elettrica viene applicata ad ogni molecola di polimero quando viene accelerata nel campo elettrico tra i due elettrodi.

I polimeri colpiranno il rivelatore, i più piccoli per primi, poi quelli più grandi. Colpiscono lo strumento in ordine di massa. Tutte le molecole polimeriche con lo stesso peso molecolare raggiungeranno il rivelatore nello stesso momento. La dimensione del picco è proporzionale al numero di molecole che colpiscono il misuratore nello stesso momento. Alla fine dell'esperimento otteniamo una serie di picchi che vengono rappresentati così:

                                                                    

Poiché il tempo necessario ad una molecola per colpire il rivelatore è proporzionale alla sua massa ciò che otteniamo è in effetti un grafico del peso molecolare sull'asse-x ed il numero di molecole con un determinato peso molecolare sull'asse-y. In questo modo otteniamo la distribuzione del peso molecolare. Un altro piccolo dettaglio che potete notare è che il peso molecolare su questo grafico aumenta da sinistra a destra. Su un grafico SEC, che vedrete più spesso, il peso molecolare aumenta da destra a sinistra. Ci sono  differenze  tra SEC e MALDI. SEC vi da una distribuzione approssimativa del peso molecolare. SEC misura il volume idrodinamico e non il peso molecolare mentre MALDI fornisce una misurazione assoluta della massa .

 

Matrix-assisted Laser Desorption/Ionization (MALDI)

 

The study of polar compounds has always been a problem for mass spectrometry. It was demonstrated  that irradiation of low-mass organic samples with a high-intensity laser pulse, produces ions that could be successfully mass analysed.  The extension of LASER/ desorption ( LD ) to the analysis of non-volatile polar biological and organic macromolecules and polymers was an important step in the development of LD. These experiments, however, revealed an upper mass limit of 5-10 kDa. The primary requirement for successful LD is that the energy transfer from the laser beam to the analyte should take place in as short a time as possible, to prevent decomposition of the thermally labile analyte molecules.  Thus the analyte is not desorbed as the intact molecule ion in any significant amount resulting in a spectrum of low-mass fragment ions only. Another major restriction of LD ionization is the short duration of the ion burst following the laser pulse. A consequence is that the technique is unsuitable for scanning analysis on sector or quadrupole instruments meaning that LD is particularly suited to TOF mass spectrometry. More recently it was discovered that the use of a matrix in LD could circumvent the restrictive mass limitations of the technique. Requirements of the matrix are that it has a strong absorbance at the laser wavelength and was of low enough mass to be sublimable. A low concentration of the analyte is uniformly dispersed throughout the solid or liquid matrix, deposited on the end of a probe or onto a metal plate and introduced into the pulsed laser beam. The low concentration of analyte used has several important advantages. The efficiency of energy transfer from the laser to the analyte (via matrix) is increased, whereas problems associated with analyte dissociation are greatly reduced. The association of the analyte molecules to form high-mass clusters is also reduced and it is believed that suitable matrices can even enhance ion formation.

The Mechanism of Ion Desorption

The mechanism of MALDI , not totally understood,   work along the following lines .

 The Formation of a 'Solid Solution'. The analyte molecules are distributed throughout the matrix so that they are completely isolated from one other. This is necessary if the matrix is to form a homogenous 'solid solution' (any liquid solvent(s) used in preparation of the solution are removed when the mixture is dried before analysis).  Matrix Excitation. Some of the laser energy incident on the solid solution is absorbed by the matrix, causing rapid vibrational excitation, bringing about localized disintegration of the solid solution, forming clusters made up of a single analyte molecule surrounded by neutral and excited matrix molecules. The matrix molecules evaporate away from these clusters to leave the excited analyte molecule.  Analyte Ionization. The analyte molecules can become ionized by simple protonation by the photo-excited matrix, leading to the formation of the typical [M+X]+ type species (where X= H, Li, Na, K, etc.). Some multiply charged species, dimers and trimers can also be formed. Negative ions are formed from reactions involving deprotonation of the analyte by the matrix to form [M-H]- and from interactions with photoelectrons to form the [M]-° radical molecular ions. These ionization reactions occur in the first tens of nanoseconds after irradiance, and within the initial desorbing matrix/analyte cloud. It is in this way that the characteristic MALDI spectra are created.

 

################################################################################################################################################################

 

MALDI melanin 0.doc

 

126. A.Selva, U.Vettori

 

  Mass Spectrometry of heterocyclic compounds

 

  Gazz.Chim.Ital. 103, 223-229, 1973

 

Four papers were  published on  fragmentation ( other terms used, explosion, laser ablation, laser disintegration ) of heterocycles compounds and their sustituents.The fragmentation pattern have been rationalized by means of high resolution mass measurements and defocused metastable ion detections.The mass spectra of seven pairs of rengiomeric heterocycles have been examined.Remarkable differences on the behaviour of positional isomers are pointed out.

 

It is interesting to note that melanin is easily fragmenteted.

MALDI is the most powerful tool for chemical structural  studies of melanins and the particles.Some difficulties occurs for reproducible results and purification of the materialThe beaviour of heterocycles compound require further investigation………………..

 

 

 

 

 

###########################################################

 

 

127. MALDI melanin 1.doc

 

 

blank

A Reassessment of the Structure of 5,6-Dihydroxyindole-2-carboxylic Acid Melanins by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

 

Rapid Comm.Mass Spectrom., 10, 204-208, 1996

 

 

Alessandra Napolitano , Alessandro Pezzella , Giuseppe Prota , Roberta Seraglia , Pietro Traldi

 

 

 

Matrix-assisted laser desorption/ionization mass spectrometry was applied to the structural investigation of synthetic melanins prepared by enzymatic or chemical oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a major biosynthetic precursor. Following optimization of the experimental conditions, spectra with a clearly distinct pattern of peaks could be obtained for all melanin samples. In contrast to the commonly held notion of melanins as high molecular weight polymers, all the pigments analyzed are mixtures of oligomer species with molecular weights in the range 500-1500 Da. Marked differences in the nature of the oligomer components were observed depending on the preparation conditions. Indeed, significant degradation of the pigment backbone, resulting probably by peroxidative fission at the catechol site of the DHICA units, was found in most of the melanin pigments, even those prepared under mild oxidation conditions. The relevance of these results to the current use of synthetic melanins as models of the natural pigments is discussed.

blank

 

The first  interesting paper on DHICA –melanin.The fact that melanins are particles without  molecular weight is well known.

 

 

 

#####################################################################

MALDI melanin 10 .doc

 

128 . Isolation and characterization of mammalian eumelanins from hair and irides.


          BBA, 1475, 295-306, 2000

 
          Novellino L, Napolitano A, Prota G.

 
A new enzymatic procedure was developed for isolation of eumelanin from black human hair which might provide a substantially intact pigment for structural characterization. Sequential digestion with protease, proteinase K and papaine in the presence of dithiothreitol afforded a pigment with a 6% w/w protein content. HPLC analysis of pyrrole acids resulting from alkaline H2O2 degradation, carboxyl content determination, and ferricyanide titration showed that the isolated pigment is made up of 5,6-dihydroxyindole (DHI)- and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units at a 6:1 ratio, exhibiting a significant degree of oxidative degradation. For comparison, a different eumelanin isolated from black bovine irides by a similar enzymatic procedure was analyzed. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the final pigment provided evidence for homologous series of DHICA oligomers, while chemical analysis allowed an estimate of 2:1 DHICA/DHI-derived units in the polymer, with a substantial proportion of intact o-diphenolic functions. Iris melanin proved able to promote the Fenton oxidation of deoxyribose while hair melanin was ineffective. Overall, these results provide, for the first time, unambiguous evidence for marked structural differences of mammalian eumelanins which may be directly related to the diversity of the sites of biosynthesis and storage, as well as to functional role of these pigments.

 

 

 

 

 

 

###########################################################.

MALDI melanin 11.doc

 

129.     Melanogenesis from 5-hydroxytryptamine, 5,6- and 5,7-dihydroxytryptamines. An in vitro study using MALDI-TOF.


        Adv.Exp.Med.Biol.467, 779-787, 1999


        Bertazzo A, Favretto D, Costa CV, Allegri G, Traldi P.

        
bertazzo@dsfarm.unipd.it

The role of tyrosinase and peroxidase in melanogenesis of 5-hydroxytryptamine, 5,6- and 5,7-dihydroxytryptamines was investigated by matrix-assisted laser desorption/ionization mass spectrometry. Each enzyme was incubated with the tryptamine derivatives and samples were drawn at various times, ultrafiltered and immediately lyophilized. The results indicated that peroxidase promotes oligomerization of 5-HT with fast kinetics but with yields lower than those achieved by tyrosinase. 5,6- and 5,7-DHT formed low molecular mass oligomers in the presence of peroxidase alone. The addition of hydrogen peroxide evidences different reactivity of the two isomers: 5,6-DHT formed immediately a black precipitate while oligomers of the molecule itself and of its oxidation products were detectable for 5,7-DHT.

 

##################################################################.

MALDI melanin 12.doc

 

130.     Application of matrix-assisted laser desorption/ionization mass spectrometry to the detection of melanins formed from Dopa and dopamine.


         Mass Spectrom.,34, 922-929, 1999


         Bertazzo A, Costa CV, Allegri G, Favretto D, Traldi P.

 
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study melanogenesis starting from Dopa and dopamine, the latter considered one of the precursors of neuromelanins. These substrates were left to react with the peroxidase – H2O2 system, which is postulated to play an important role in melanin biosynthesis. Samples were prepared by ultrafiltering the substrate - enzyme solution after 30, 60, 120, 240 and 360 min of reaction and aliquots were immediately lyophilized. The reaction of dopamine with peroxidase - H2O2 favoured the formation of dopamine oligomers up to octamers. In contrast, the action of either peroxidase or H2O2 alone, studied for comparison, did not lead to melanin production and only dimeric and trimeric species were observed. Also for Dopa, analogous results were obtained in the presence of either peroxidase or H2O2 alone, without melaninformatio Conversely, Dopa with the peroxidase - H2O2 system led to the formation of a black precipitate after 120 min of reaction, and oligomers of 5,6-dihydroxyindole (DHI), an intermediate of melanogenesis, were detected, together with products of further oxidation. Faster kinetics were observwhen Dopa was treated with tyrosinase, the enzyme catalysing the oligomerization of tyrosine to melanins, leading to the formation mainly of DHI oligomers.

###########################################################.

MALDI melanin 13.doc

 

  131. Mechanism of selective incorporation of the melanoma seeker 2-thiouracil into growing melanin.


J.Med.Chem. 39, 5192-5201, 1996


Napolitano A, Palumbo A, d'Ischia M, Prota G.

The mechanism of selective incorporation of 2-thiouracil (TU), a highly specific

  melanoma seeker, into growing melanins was investigated both in vitro and in vivo. Methods used included direct analysis of the melanins, by evaluation of the absorption at 350 nm (A350) and chemical degradation coupled with HPLC quantitation of pigment makers, i.e., pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), as well as biosynthetic experiments involving tyrosinase-catalyzed oxidation of DOPA, 5,6-dihydroxyindole (DHI), and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Injection of radiolabeled TU into melanoma-bearing mice resulted in a rapid incorporation of the drug into the tumor pigment, with a substantial decrease in A350 and in PTCA yields. Similar changes in the absorption properties were observed in biosynthetic melanins prepared in the presence of TU, whereas the yields of PTCA and PDCA varied depending on the pigment precursor used. When incubated with DOPA in the presence of tyrosinase, TU profoundly modified the normal course of melanogenesis, favoring formation of a complex mixture of addition products consisting mainly of 6-S-thiouracil-DOPA as well as DHI-TU adducts. The latter were obtained in larger amounts by enzymatic oxidation of DHI in the presence of TU and were identified as the 3- and 2-substituted adducts 1 and 2, the dimer 3, and the trimer 4. Similar reactions carried out on DHICA yielded the 4-substituted adduct 5, the dimer 6, and the trimer 7. A new mechanistic scheme for the incorporation of TU into growing melanin is proposed, which envisages nucleophilic attack of the thioureylene moiety of TU to transient quinonoid intermediates in the melanin pathway, chiefly dopaquinone and 5,6-indolequinones, followed by entrainment of the resulting adducts

 

 

 

 

 

 

 

#########################################################.

MALDI melanin 14.doc

 

132  Nicotine and cotinine adducts of a melanin intermediate demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.


         Chem.Res.Toicol., 14, 275-279, 2001


          Dehn DL, Claffey DJ, Duncan MW, Ruth JA.

 Pigmentation is a major factor in the incorporation of many drugs into hair. In an attempt to elucidate potential mechanisms of drug-melanin interaction, melanin was synthesized in vitro in the presence of nicotine, which we have shown to have a substantial interaction with melanin, and cotinine, a primary nicotine metabolite. L-DOPA, a precursor of eumelanin, was oxidized and oligomerized with tyrosinase. Nicotine, cotinine, and/or their deuterated analogues were added to the oligomerization reaction mixture in a 10:1 L-DOPA:drug ratio. A black precipitate formed within 60 min. Aliquots were removed from the incubation mixture at 60, 120, and 360 min. MALDI-TOF MS determinations were carried out on each sample to provide a mean and standard error for the masses of interest. Internal calibration allowed accurate mass measurement of the products. A careful comparison of the spectra of samples prepared both with and without drug indicated the presence of masses corresponding to the protonated drug, melanin oligomers, and nicotine or continine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). Additional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and observing that the masses shifted accordingly. Structures of the adducts were further confirmed by select ion gating and postsource decay analysis

 

 

 

-----------------------------------------------------------------------------------------------------------

#######################################################.

MALDI melanin 15.doc

 

 

  133. Effects of ultraviolet irradiation on melanogenesis from tyrosine, Dopa and dopamine: a matrix-assisted laser desorption/ionization mass spectrometric study.

Rapid Comm.. Mass Spectrom. 14, 1862, 2000


Bertazzo A, Favretto D, Costa CV, Allegri G, Traldi P.

 Matrix-assisted laser desorption/ionization (MALDI) mas
s spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

####################################################################.

 

MALDI melanin 2.doc

 

 

 

  134.  Structural Analysis of Synthetic Melanins from 5,6-Dihydroxyindole by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

 

Rapid Comm. Mass Spectrom. 10, 468-472, 1996

 

Alessandra Napolitano , Alessandro Pezzella , Giuseppe Prota , Roberta Seraglia , Pietro Traldi 

 

 

 

Despite extensive investigation, the structure of melanins, the major determinants of skin colour differences in man and other mammals, is still poorly defined, mainly because of the unfavourable properties of the materials. In this study, analysis of model pigments prepared by enzymically- or chemically-induced oxidative polymerization of 5,6-dihydroxyindole (DHI), the ultimate biosynthetic precursor, was investigated by means of matrix-assisted laser desorption/ionization mass spectrometry. All DHI melanins exhibited quite distinct pattern of ionic species of low m/z ranging from 500 to 1500, none of which corresponded to intact DHI oligomers. Analysis of the molecular weights and mass differences between the oligomer species provided evidence for a significant breakdown of the pigment backbone by peroxidative fission of the indole units with concomitant decarboxylation and oxygenation reactions. These processes, which take place to a different extent depending on the preparation conditions, are possibly initiated by hydrogen peroxide, either added as the oxidant or slowly generated in the reaction medium. In support of this, melanin samples prepared by tyrosinase oxidation in the presence of catalase comprised, as major components, intact DHI oligomers up to hexamers. Overall, these results offer a new picture of the structure of DHI melanins which may significantly contribute to the understanding of the high degree of molecular heterogeneity of the natural pigments.

 

 

 

 

#################################################################.

MALDI melanin 20.doc

 

135.   Amphetamine adducts of melanin intermediates demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

 

Chem.Res.Toxicol., 14, 1339-1344, 2001

 

Claffey DJ, Ruth JA.

 

 

The use of hair as a matrix for the determination of a history of drug abuse is becoming increasingly widespread. Melanin has been shown to play a key role in the incorporation of drugs in hair. The mechanism of this incorporation and the nature of the interaction remains poorly understood. Cationic drugs, such as amphetamine, are thought to be ionically bound to melanin; however, their inextricability has led to the suggestion that they may be covalently bound to a great degree. Identification of covalent adducts remains elusive due to the insoluble polymeric nature of melanin. We succeeded in identifying several such adducts by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) analysis of the products of in vitro synthesis of melanin in the presence of amphetamine. Amphetamine was incubated with L-DOPA and mushroom tyrosinase under a stream of oxygen. After 1 h, a signal at m/z 281.1324 (n = 1, R = H) was observed. After 2 h, the major adduct mass visible in the spectrum was at m/z 470.1074. This appeared to be derived from the mono-decarboxylation of a minor adduct at m/z 514.1245 (n = 2, R = CO(2)H). A totally decarboxylated adduct was also observed at m/z 426.1448 (n = 2, R = H). These were identified as amphetamine adducts of indole quinones. Corroboration of their identity was obtained by observing the mass shifts with deuterated L-DOPA and amphetamine analogues. Accurate mass measurements using the reflectron mode of the MS showed that the smaller adduct was within 14 ppm, and the larger adducts were within 70 ppm of their theoretical monoisotopic masses. Postsource decay experiments agreed with our structural assignments.

 

 

 

 

 

####################################################################.

MALDI melanin 3.doc

 

 

 

 

 

 

      136.  Identification of Partially Degraded Oligomers of 5,6-Dihydroxyindole-2-carboxylic Aci in Sepia Melanin by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

 

Alessandro Pezzella , Alessandra Napolitano , Marco d'Ischia , Giuseppe Prota , Roberta Seraglia , Pietro Traldi

 

Rapid Comm.Mass Spectr. 11,368-372 (1997)

 

 

Despite extensive efforts over more than half a century, the direct structural investigation of natural melanins has, so far, been largely unsuccessful, because of the adverse physical and chemical properties of these pigments. In the present study, we applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the direct analysis of fresh melanin samples from the cuttlefish Sepia officinalis, and succeeded in identifying clearly distinct patterns of low molecular weight ionic species, ranging from 450 to 1200 Da. Detailed analysis of the molecular weights and mass differences between the major species, aided by comparison with MALDI-MS spectra of synthetic melanins, allowed formulation of the major components as oligomers of 5,6-dihydroxyindole-2-carboxylic acid partially degraded by peroxidative fission of the o-diphenol moieties with concomitant decarboxylation and oxygenation reactions. These results provide the first direct insight into the structure of Sepia melanin and confirm the unique value of MALDI-MS techniques for the investigation of highly complex and heterogeneous polymers such as melanins.

 

 

The first example of eumelanin fragmentation.The authors suggest DHICA as the major precursor of sepiomelanin.

 

blank

blank

 

 

 

-----------------------------------------------------------------------------------------------------

 

#######################################################################.

MALDI melanin 4.doc

 

 

     137. Enzymatic oligomerization of tyrosine by tyrosinase and peroxidase studied by matrix-assisted laser desorption/ionization mass spectrometry

 

Rapid Comm.Mass Spectrom., 13, 542-547, 1999

 

 

Antonella Bertazzo , Carlo V.L. Costa , Graziella Allegri , Marta Schiavolin , Donata Favretto , Pietro Traldi

 

 

The role of tyrosinase in melanogenesis starting from tyrosine, considered the physiological melanin precursor, was compared with that of peroxidase /H2O2 by matrix-assisted laser desorption/ionization mass spectrometry. Samples were prepared by ultrafiltering the tyrosine-enzyme solution at various reaction times, and were then immediately lyophilized. In parallel, samples obtained by the action of peroxidase or H2O2 alone were analyzed and compared. Samples originating from the action of tyrosinase on tyrosine revealed the presence of oligomers of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and DHI-DHICA, and their molecular masses increased with increasing reaction times up to octamer levels. When the reaction was carried out in the presence of the peroxidase/H2O2 system, a completely different oligomeric pattern was revealed in the ultrafiltered samples. In fact, the species detected at various times were not due to DHI oligomers, but showed a tyrosine-based skeleton.

 

blank

 

 

 

 

 

 

#########################################################.

MALDI melanin 5.doc

 

  138.  A.Bertazzo, C.Costa, G.Allegri, R.Seraglia, P.Traldi 

 

 Biosynthesis of melanin from Dopamine. An investigation of early oligomerization products

 

 Rapid Comm., Mass Spectrom., Vol. 9, 634-640,  1995

 

 

Matrix – assisted laser desorption/ionisation (MALDI )and fast – atom bombardment (FAB)mass spectrometry  experiments were applied to the study of the early stages of the early stages of the oligomerization  reaction of dopamine with mushroom tyrosinase. Ultrafiltration was  employed to remove the enzyme at various reaction times, to prevent possible attachment of the protein to the highly reactive intermediates. Two sets of five samples each, obtained  at different reaction times, in one case immediately  lyophilised and in the other left to react under an oxygen stream for 24h before lyophilization, were compared. FAB showed  the presence of various species and of these, that at m/z 305 increased in abundance with reaction time in the immediately lyophilised set of samples only. Accurate  mass measurements and tandem mass spectrometric experiments indicated the structure of  a dopamine protonated dimer for this ion.  MALDI measurements  showed that all samples were composed of clusters  of oligomers  differing in degree of oligomerization. Oligomerization increases with reaction time, resulting in the formation of  species at 2643- 2911 Da. These  clusters in turn were formed of species with a different  degree of  oxidation, detected in both sets of samples.

 

 

 

########################################################################

######################################################.

MALDI melanin 6.doc

 

 

139. C.Kroesche, M.G.Peter 

 

  Detection of Melanochromes by MALDI-TOF Mass Spectrometry    

 

Tetrahedron 52, 3947-3952, ( 1996 )

 

 

 

Melanin formation from dopamine, DOPA, DHICA, was analysed by means of matrix-assisted laser desorption mass spectrometry. Oligomers of dihydroxyindoles, melanochromes up to DP  11  were deteced.Increments of 16 mass units provide evidence for the presence of trihydroxyindoles units.The results indicate that polymerisation of dihydroxyindoles towards melanin occurs by sequential coupling of monomers with concomitant oxygenation

 

 

 

 Secondo Peter la polimerizzazione di DA (dopammina ), e DHI procede per unità integre . Si nota per la prima volta nel corso della melanogenesi ossidrilazione delle unità.*

La polimerizzazione del DHICA  per autossidazione porta ad oligomeri da 3 a 5.La reazione procede  con ossidrilazione e decarbossilazione ( formazione di DHI ) che viene indicato nella letteratura come originatesi dal dopacromo..

 Osservato ( DHI )2 - ( DHICA )3

La polimerizzazione di DOPA / tirosinasi porta a miscele eterogenee in cui si può riconoscere sia DHI che DHICA.

 

 Nello importante lavoro di Peter si dice che l’addizione di acqua sia un fenomeno mai riferito prima. In effetti il fenomeno  è conosciuto da molto tempo ed è facilmente rivelabile con  l’analisi.

Il quadro chimico della melanogenesi di Peter è molto diverso da quello al quale giunge Prota (127), (134), (136)

 

 Hearing (70 )   in un lavoro del 1980,  che la comunità scientifica ha sottovalutato,   dimostra che non vi sono nella tirosina-melanina  o nella DOPA-melanina unità carbossilate e cioè unità di tirosina, DOPA, ciclodopa, dopacromo, DHICA.

  Allegri (138 ) e  Bolognese (  Lettera MALDI Link 12 pag 12)   non trovano nei loro studi  MALDI unità degradate.

 

 

* Trihydroxy- derivatives of cathecol  are also  formed during  allomelanogenesis. ( humic acid,Ustilago melanin, catechol-melanin f) as showed by Dawson ( Ann.New York Academy of Sciences,100, 937-948, 1963 .

 

################################################################################################################################################################

 

 

140. C.R.Dawson, W.B.Tarpley

 

 

On the pathway of the catechol-tyrosinase  reaction

 

Acc.New York Acad.Sci., 100, 937-948, 1963.

 

 

 

141.D.G.Graham, P.W.Jeffs

 

The role of trihydroxyphenylalanine in  melanin biosynthesis

 

 

J. Biol.Chem. 252, 5729-5734, 1977

 

--------------------------------------------------------------------------------------------------------------------

 

 

142. The first who observed the formation  of 6-hidroxydopa where Lissitzky and Rolland, Nature, 193, 881, 1962

 

------------------------------------------------------------------------------------------------------------------------

 

143. C.Hansson, H.Rorsman, E.Rosengren

 

5-Hidroxydopa a new compound in the Raper-Mason scheme of melanogenesis

 

Acta Dermatovener. 60, 281, 1980 ; 1981 ; G.Agrup,H.Rorsman, E.Rosengren 1982

 

---------------------------------------------------------------------------------------------------------------

 

 

 

144 . 5-Hidroxydopa was not considered an intermediate by :

 

G.Prota   Melanins  and melanogenesis  AP, San Diego ,1992, pag.49.

 

R.F.Chapman, A.Percival, G.A.Swan,  J.Chem.Soc. 1664,1970

 G.A.Swan, J.C.S.Perkin I, 339, 1976. 

 

###################################################################.

-------------------------------------------------------------------------------------------------

MALDI melanin 7.doc

 

145.     Melanogenesis by tyrosinase action on 3,4-dihydroxyphenylalanine (DOPA) in the presence of polyethylene glycol: a matrix-assisted laser desorption/ionization mass spectrometric investigation.



         Rapid Comm.Mass Spectrom., 15, 1061-1067, 2001

         Bertazzo A, Vogliardi S, Favretto D, Costa CV, Allegri G, Traldi P.

 

The enzymatic reaction between DOPA and tyrosinase, the enzyme considered to be responsible for melanogenesis, was carried out in the presence of polyethylene glycol (PEG). This choice was made in order to increase the solubility of melanins, since these polymers are highly insoluble. The reaction mixtures were sampled at different times, immediately ultrafiltered to remove the enzyme, lyophilized, and analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The results were very different from those obtained in the absence of PEG. Only a  few oligomers of dihydroxyindole (DHI) and dihydroxyindole-2-carboxylic acid (DHICA) were detected in low abundances, whereas newspecies originating from reaction of PEG with species belonging to the Raper-Mason pattern appeared. The results show that, in the presence of PEG, tyrosinase-catalyzed oligomerization of DOPA exhibits kinetics slower than those observed in the absence of the polymer. However, melanogenesis still takes place in the presence of PEG, as indicated by the formation of black pigments and by the detection of DHI and DHICA oligomers, considered to be the first intermediates in melanin formation

 

In questo interessante lavoro  mentre si dimostra l’importanza della tirosinasi da una altra parte risulta sempre più evidente come sia non valido lo studio della melanogenesi con lo schema di RaperiLa presenza carbossili provenienti dalla rottura di unità catecoliche rende poco probabile che vi siano in natura delle DHICA melanine.

 

---------------------------------------------------------------------------------------------------------

 

################################################################.

MALDI melanin 8T.doc

 

 

146                        doppio col 132  

 

 

 

###########################################################################.

 

MALDI melanin 10 .doc

 

   147                doppio col    133    

 

 

 

 

148          Laser desorption mass spectrometry in the study of natural and synthetic melanins.III.Human hair melanins

 

Eur.J.Mass Spectrom, 1, 305-312, 1995

 

A.Bertazzo, C.Costa, G.Allegri

 

 

Four samples of natural melanin extracted from red, fair, black and brown human head hair were studied by both laser desorption/ionisation ( LDI ) and matrix- assisted  laser desorption/ionisation ( MALDI ) methods . Clear difference were found among the LDI and MALDI mass spectra of the four melanins. In red hair melanin, there was a major peak  at m/z 842 attributed to an oligomeric cluster, together with lower-intensity peaks in the range 4000-5000 Da.In fair hair melanin,only species with molecular masses from 300 to 600 Da were evidenced.In black hair melanin, clusters up to 25000 Da were detectable, and in brown hair  melanin there were two main oligomeric cluster in the ranges 600-900 Da and 3000-5000 Da, respectively, and a large oligomer  distribution from 11000 to 16000 Da . It is concluded that there is an increase in relative molecular masses of melanins on passing from fair to black hair, suggesting that in dark hair the colour is related to melanins with a higher degree of polymerisation

 

 

 

Inizio modulo

 

 

 

 

 

 

 

 

 

NANOSCIENCES

 

 

 

 

 

################################################################################.

Morphology of allomelanin particles was known since 1980.

 

 

149.  D.M.Rast, H.Stuessi, H.Hegnauer    Melanins    in Fungal Spore  :  morphogenetic controls

( Proc. Int. Fungal Spore Symp. 1980  )

 

Eds. Turian,Hohl

Academic London   1981

 

 

#########################################################################.

The first steps of the particle

 

 

150.  Melanin standard method : particle description

 

Pigment Cell Research  5, 133-142, 1992

 

L.Zeise, B.L.Murr, M.R.Chedekel

 

 

 

  151.  Bio-analytical studies of eumelanins.I.Characterization of melanin the particle.

 

Pigment Cell Research Suppl.2, 48-53, 1992

 

         L.Zeise, R.B.Addison, M,R.Chedekel

 

 

#####################################################.

 

152       Morphology of cultured human epidermal melanocytes observed by atomic force microscopy.

 

        Pigment cell res.  17, 62-65, 2004

         Zhang RZ,
Zhu WY , Xia MY, Feng Y.

 
The objective of this study was to image the surface structure of cultured human epidermal melanocytes using atomic force microscopy (AFM). Epidermis obtained from human foreskins was treated with 0.5% dispase. Cell suspensions of the epidermis were prepared and seeded in six-well plates, in which sheets of mica had been placed. Samples for AFM were fixed on mica and scanning AFM images were captured by contacting and tapping modes operated under normal atmospheric pressure and temperature. Human epidermal melanocytes exhibited rounded, oval, triangular or quadrangular perikarya from which eight to 10 thick dendrites arose. These dendrites first bifurcated near the soma and then divided profusely into daughter branches, which spread out in all directions. We observed string-like long thin projections, growth cones and shorter thicker projections, which arose from the dendritic shafts, in which groups of melanosomes were arrayed. In addition to such structures, the most striking feature was the presence of filopodia arising from the melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes. The termini of dendrites formed unbranched terminal protrusions (approximately 1500-2000 nm wide) consisting of two to three melanosomes wrapped in an arc, with their filopodia extending outwards. The tips of these structures also appeared to be squeezed and finally pinched off by the melanocyte to form a pouch filled with numerous melanosomes. We conclude that secondary and tertiary branches and subordinate branches might take part in transferring melanosomes into keratinocytes in addition to the transfer through the tips of the dendritic shafts. The melanin granules were expelled by exocytosis.

 

------------------------------------------------------------------------------

 

  ##########################################################################.
  153. 
Isolation and biophysical studies of natural eumelanins: applications of imaging technologies and ultrafast spectroscopy.



Pigment Cell Res. 16, 608-618, 2003

Liu Y, Simon JD.



 
The major pigments found in the skin, hair, and eyes of humans and other animals are melanins. Despite significant research efforts, the current understanding of the molecular structure of melanins, the assembly of the pigment within its organelle, and the structural consequences of the association of melanins with protein and metal cations is limited. Likewise, a detailed understanding of the photochemical and photophysical properties of melanins has remained elusive. Many types of melanins have been studied to date, including natural and synthetic model pigments. Such studies are often contradictory and to some extent the diversity of systems studied may have detracted from the development of a basic understanding of the structure and function of the natural pigment. Advances in the understanding of the structure and function of melanins require careful characterization of the pigments examined so as to assure the data obtained may be relevant to the properties of the pigment in vivo. To address this issue, herein the influence of isolation procedures on the resulting structure of the pigment is examined. Sections describing the applications of new technologies to the study of melanins follow this. Advanced imaging technologies such as scanning probe microscopies are providing new insights into the morphology of the pigment assembly. Recent photochemical studies on photoreduction of cytochrome c by different mass fraction of sonicated natural melanins reveal that the photogeneration of reactive oxygen species (ROS) depends upon aggregation of melanin. Specifically, aggregation mitigates ROS photoproduction by UV-excitation, suggesting the integrity of melanosomes in tissue may play an important role in the balance between the photoprotective and photodamaging behaviors attributed to melanins. Ultrafast laser spectroscopy studies of melanins are providing insights into the time scales and mechanisms by which melanin dissipates absorbed light energy.

Use of pure and stabilized  samples of granules is recommended

          

 

 

 

############################################################.

154.  Ultrafast energy transfer from bound tetra(4-N,N,N,N-trimethylanilinium)porphyrin to synthetic dopa and cysteinyldopa melanins.



Photochem.Photobiol., 77, 1-4, 2003


Ye T, Simon JD, Sarna T.

 
The binding of tetra(4-N,N,N,N-trimethylanilinium)porphyrin (TAP) to melanins quenches the porphyrin emission. Time-resolved femtosecond absorption spectroscopy reveals that the mechanism behind this quenching is ultrafast nonradiative energy transfer ((tau)ET < 100 fs) from electronically excited TAP to melanin. Similar dynamics are observed for both dopa and cysteinyldopa melanins. Steady-state emission studies demonstrate that the emission from melanin increases upon excitation of bound TAP, thereby confirming that rapid energy transfer occurs. These results are consistent with previous photoacoustic studies, which revealed that the TAP-melanin complex behaves like a supermolecular system liberating heat as a whole.

----------------------------------------------------------------------------------

######################################################################.
155. 
Unlocking the molecular structure of fungal melanin using 13C biosynthetic labeling and solid-state NMR.


Biochemistry 42, 8105-8109


Tian S, Garcia-Rivera J, Yan B, Casadevall A, Stark RE.

 
Melanins are enigmatic pigments found in all biological kingdoms that are associated with a variety of functions, including microbial virulence. Despite being ubiquitous in nature, melanin pigments have long resisted atomic-level structural examination because of their insolubility and amorphous organization. Cryptococcus neoformans is a human pathogenic fungus that melanizes only when provided with exogenous substrate, thus offering a unique system for exploring questions related to melanin structure at the molecular level. We have exploited the requirement for exogenous substrate in melanin synthesis as well as the capabilities of high-resolution solid-state nuclear magnetic resonance (NMR) to establish the predominantly aliphatic composition of l-dopa melanin and to introduce 13C labels that permit the identification of proximal carbons in the developing biopolymer. By swelling solid melanin samples in organic solvents and using two-dimensional heteronuclear NMR in conjunction with magic-angle spinning, we have identified chemical bonding patterns typical of alkane, alkene, alcohol, ketone, ester, and indole functional groups. These findings demonstrate the feasibility of a novel approach to determining the structure of melanin using metabolic labeling and NMR spectroscopy.

############################################################.

 

 

  156.  Reconstituted 3-dimensional human skin of various ethnic origins as an invitro model for studies of pigmentation.


Anal.Biochem., 318, 260-269


Yoon TJ, Lei TC, Yamaguchi Y, Batzer J, Wolber R, Hearing VJ.

 Reconstituted 3-dimensional human skin equivalents containing melanocytes and keratinocytes on an artificial dermal substitute are gaining popularity for studies of skin metabolism because they exhibit morphological and growth characteristics similar to human epidermis. In this study, we show that such a pigmented epidermis model can be used to assess the regulation of pigmentation by known melanogenic compounds. In monolayers or in melanocyte-keratinocyte co-cultures, melanocyte-keratinocyte interactions are missing or are spatially limited. The commercial skin equivalents used in this study were derived from epidermal cells obtained from donors of three different ethnic origins (African- American, Asian, and Caucasian), and they reflect those distinct skin phenotypes. We used these pigmented human epidermis models to test compounds for potential effects on pigmentation in a more physiologically relevant context, which allows further characterization and validation of interesting melanogenic factors. We used known melanogenic stimulators (alpha-melanocyte-stimulating hormone and 3,4-dihydroxyphenylalanine) and inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and examined their effects on the production of melanin and its distribution in upper layers of the skin. Our studies indicate that commercial skin equivalents provide a convenient and cost-effective alternative to animal testing for evaluating the regulation of mammalian pigmentation by melanogenic factors and for elucidating their mechanisms of action.


-----------------------------------------------------------------------------------------------------

 

################################################################.

---------------------------------------------------------------------------------

 

157.   The effect of preparation procedures on the morphology of melanin from the ink sac of Sepia officinalis.


         
Pigment Cell Res., 16, 72-80, 2003


          Liu Y, Simon JD.

 
The structure of melanin extracted from the ink sac of the cuttlefish Sepia officinalis was examined for different methods of isolation and purification of the pigment. Scanning electron microscopy (SEM) images of Sepia eumelanin prepared by different procedures establish that multi-microm-sized aggregates reported by previous workers are generated by their sample preparation, and that the dominant constituents of Sepia melanin are approximately 150 nm spherical granules. Brunauer-Emmett-Teller (BET) measurements reveal that Sepia eumelanin from Sigma (prepared by spray drying the pigment) has a surface area of 14.3 m2/g. Pigment extracted directly from the fresh ink sac and then freeze-dried has a surface area of 21.5 m2/g, while CO2-supercritically dried has a surface area of 37.5 m2/g. This is consistent with SEM images showing that the process of freeze-drying produces aggregates, but to a lesser extent than spray drying. Supercritical drying of the sample produces suspensions of the individual approximately 150 nm granule, which is more reflective of the natural pigment. Brunauer-Emmett-Teller surface area analysis and Barrett-Joyner-Halenda (BJH) pore volume analysis indicate that the surface of the granules is not smooth and the interior of the granules is not porous, but rather the aggregates of granules are porous. Ultra-high resolution SEM and atomic force microscopy (AFM) images show the granules are easily deformed and are comprised of smaller constituents. De-aggregation of the granules by sonication and ultra-filtration reveal a range of structures depending on the pore size of the membrane used. The implications of these results on quantifying photochemical properties and kinetic reaction rate constants of melanin are discussed.

 

 

Morphology results  may change with composition of the samples (sepiomelanin or sepiomelanic acid,  melanosomes, premelanosomes, degraded particles ) . Determination  of conductivity,IR,NMR ,MALDI of different aggregate structures from pure and stabilized granules may be interesting.

 

.
####################################################################.

158.  Computer vision and digital imaging technology in melanoma detection.


Semin.Oncol., 29, 308-327, 2002


Voigt H, Classen R.

 
With today's treatment options, melanoma cure rates can be improved only if the diagnosis is made early enough to allow for curative surgery. Since clinical signs of malignancy in a pigmented lesion are often ambiguous and even dermatology experts may misdiagnose melanoma, diagnostic tools and procedures have been developed to assist the clinician in the diagnostic workup. Epiluminescence microscopy or dermatoscopy is widely used to inspect the melanin reticulum at the epidermo-dermal junction zone for signs indicative of early tumor growth. With the help of computer technology, digital dermatoscopy systems have entered the diagnostic arena capable of accurately assessing skin surface features modeled along the ABCD criteria already used for clinical assessment of pigmented skin lesions. Today's technically refined computer-based systems provide sophisticated functionalities for automated feature extraction and lesion assessment for quantitative analysis, and may also be used for education and training purposes.

-----------------------------------------------------------------------------------------------

  #########################################################################.
159. 
Morphological studies on microfilaments and their organizing center in killifish (Fundulus heteroclitus L.) melanophores.


Pigment Cell Res.,15, 298-304, 2002


Kimler VA, Palazzolo KL, Anne P, Haddad MM, Lee JB, Harkins C, Vallarapu B, Taylor JD.

 
Fish chromatophores serve as excellent study models for cytoskeleton-dependent organelle translocations because the distribution of pigmentary organelles can be observed against a time frame by microscopy. In this study the distribution of microfilaments along with microtubules in cultured melanophores of the killifish (Fundulus heteroclitus Linneaus) are examined using whole-cell transmission electron microscopy (WCTEM), fluorescence, and laser scanning confocal microscopy. Dispersing, dispersed, aggregating and aggregated states of pigment are induced by adding either caffeine (for dispersion) or epinephrine (for aggregation) to the cells in a standard culture medium. The cells that exhibited a random melanosome distribution in the standard culture media without these two reagents, served as the control. The results indicate that: (i) a structure considered to be the actin-filament organizing center (AFOC) is in close proximity to the microtubule-organizing center (MTOC); (ii) the radial layout of microfilaments remains similar over four physiological states of pigmentary response with the exception of epinephrine-aggregated pigment, in which the aggregate blocks the viewing of the AFOC and central microfilament rays, yet radial microfilaments, whether central and/or peripheral, are apparent in all physiological states of distribution; and (iii) microfilaments serve, together with microtubules, as scaffolding for melanosomes which migrate in bi-directional rows on cross-bridges, thus shedding light on the mechanisms for orderly melanosome translocations in a structural continuum.

 

 

################################################################################.

160.  Synthesis of optically active tetrameric melanin intermediates by oxidation of melanogenic precursor 5,6-dihydroxy-2-carboxylic acid under biomimetic conditions

 

Tetrahedron Asymmetry, 14, 1133-1140, 2003

 

A.Pezzella, G.Di Vogna, G.Prota

 

 

Steroespecificity in particle construction

-----------------------------------------------------------------------------------

###################################################################

-------------------------------------------------------------------------------------------------------

161.    Self assembly of melanin studied by laser light scattering

 

Biophysical Chemistry 73, 227-239, 1998

 

M.G.Bridelli

 

 

The problem  of characterizing melanins was approached  by means of light scattering techniques,static and dynamic. The static technique allowed us to identify the macromolecular properties  (MW) and  ( R2g1/2 ) of melanin extracted from sepia ink sac and of tho synthetic analogues : DOPA-melanin obtained by autoxidation and by enzymatic oxidation by tyrosinase. By dynamic light scattering ( DLS )  the hydrodynamic radius Rh  was measured to monitor the temporal behaviour of the polymerisation and aggregation processes and Rh variation by changing the chemical constraints of the polymerisation medium, such as pH and ionic strength. The fractal dimension ( d ) of the aggregates of melanin, both natural and synthetic, in the part only recognized during the aggregation of the synthetic  one by lowering the pH of the medium,was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters ( group ) that are spherical,not largely hydrated and self-assembled, following a reaction limited aggregation kinetics ( d=2.38).

 

 

#####################################################

------------------------------------------------------------------------------------------

 

162.    A model for interfacial electron transfer on colloidal melanin

 

J.Photochem.Photobiol.B, 30, 119-123, 1999

 

P.R.Crippa,S.Michelini

 

Though melanins are involved in photochemical reactions ( mainly of oxido-reductive type ) in vitro and this activity is supposed to have biological implications, no satisfactory model of the reaction kinetics has so far been proposed.The main difficulty arise from the particulate structure of the insoluble melanins and the consequent necessity to describe their reactivity in the framework of heterogeneous chemistry, i.e., at the solid-liquid interface. Our paper presents a simplified model of the monoelectronic reduction reaction of dioxygen, based on well established experimental facts and some reasonable assumptions 1) surface adsorption of O2 on colloidal melanins can be described by a Langmuir isotherm  2)  the kinetics of superoxide formation are photo-dependent and include an interfacial electron-transfer process 3)  the photochemical behaviour of the single melanin granule can be described in terms of the electronic properties of amorphous semiconductor particles. Some satisfactory comparisons with experimental data and calculated values of the kinetic constants for the process are presented and discussed.

 

 

 

######################################################################.

 

163.      Ultrastructural organization of eumelanin from Sepia officinalis measured by Atomic Force  Microscopy

 

Biochemistry, 40, 13353-13360, 2001

 

C.M.R.Clancy, J.D.Simon

 

Atomic force microscopy AFM is used to investigate the structural organization of  eumelanin isolated from the ink  sacs of the cuttlefish Sepia officinalis Deposits of eumelanin  on mica reveal a range of structures.The most prevalent structure is an aggregate comprised of particles with diameters of 100-200  nm. This morphology is consistent with  published SEM images of intact granules .Mechanical manipulation of these structures using the AFM  tip show that these particles,while stable, are not a fundamental structural unit but are an aggregate of smaller constituent.Images of the bulk pigments also reveal the presence of filament structures that have an average height and width of circa 5 nm and tens of nanometers,respectively.Taken along with recent X-ray scattering and mass spectrometry experiments, the data provides strong supporting evidence for the conclusion that eumelanin is comprised  of small oligomeric units and that the structural morphology observed in imaging experiments reflects aggregation of these oligomeric molecules On the basis of the types of structures observed in the AFM  images, a model is proposed for the assembly of the macroscopic pigment.The diversity of functions attributed to melanin in the literature is proposed to result from the  heterogeneity of aggregate structures.

 

 

 

Samples formed of pure and stabilized granules of BCM  should be used in AFM experiments

 

 

 

 

 

#####################################################################.

 

164 .   A hierarchical self-assembly of Eumelanin 

 

          J.Phys.Chem B, 104, 7871-7873, 2000

 

 

          C.M.R.Clancy, J.B.Nofsinger, R.K.Hanks, J.D.Simon

 

 

Atomic force microscopy is used to examine the structure of a natural eumelanin isolated from the ink sacs of cuttlefish Sepia officinalis . The experimental data presented clearly show that the 100-200 nm spherical eumelanin particles imaged previously by SEM are not a fundamental structural unit.While these spherical particles are stable structures, as is evidenced by their cohesiveness under mechanical stress,the AFM imges reveal that these particles are composed of smaller constituents.Taking recent scattering and mass spectrometry results into consideration, we conclude that the self assembly of Sepia eumelanin is a hierarchical process with small units assembling inti hundred-nanometer structures, which then aggregate to form the morphology of the macroscopic pigment,

 

 

 

Pure granules of BCM are recommended in AFM experiments

 

################################################################################

 
164 a .     
PP-13 Metal ions complexation with melanin.

Liu Y, Kempf V, Samokhvalov A, Simon JD.

Pigment Cell Research, 16, 597-603, 2003

 
Melanin isolated from ink sac of sepia officinalis was dispersed in different concentrations of metal salt solutions (CaCl2, FeCl3, CuCl2, ZnCl2, all >99.995% pure, Aldrich) ranging from 1 microM to 10 mM. The melanin was separated from the solution by centrifugation, and the solid was washed three times with water. Then the solid was lyophilized. The metal content (Ca, Mg, Fe, Cu, and Zn) of each dried sample and the first supernatant were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The data indicates that one can enrich the metal ion contents in melanin by exposing melanin to the metal salt solutions. However, the metal contents in melanin are inter-related. For example, Ca and Mg are released when the heavy metal contents increase, but not the reverse, i.e. Ca and Mg can not displace any other heavy metals. Melanin has a much stronger binding to Fe and Cu than Ca. Fe can also displace Zn in melanin but not Cu. More over, at saturation adsorption melanin binds more Fe than any other metals, which replace the total amount of Ca and Mg, approximately 2 times of Cu and 3 times of Zn, i.e. the binding capacity of melanin to the metals has a trend of Fe>Ca approximately Mg>Cu>Zn. The number of Fe binding sites is more than the sum of Cu and Zn. Thus Fe binding sites are less selective, and the Cu and Zn have more coordination numbers to melanin. The chemical nature of the metal binding site is investigated by IR spectroscopy and Raman Spectroscopy. The fact that the Ca and Mg are displaced by any heavy metals ions adsorbed is consistent with the role of melanin as metal ion exchanger, and thus also consistent with the role of melanin as a regulator for Ca homostasis. The protection roles of melanin as sequester of toxic heavy metal ions and Ca homostasis regulation are related.

PMID: 12950814 [PubMed - as supplied by publisher]


 

 

####################################################################.

 

PHYSICAL METHODS  UV, IR ,  13C NMR,  EPR, X-rays

Under construction

 

NMR

 

################################################################################

 

165.  Solid-state cross-polarization magic angle spinning 13C and 15N  NMR characterization of Sepia melanin, Sepia melanin free acid and Human hair melanin in comparison with several model compounds

              B.B.Adhyaru, N.G.Akhmedov, A.R.Katritzky, C.R.Bowers

             Magn.Reson.Chem.  41, 466-474, 2003

 

This paper  presents the high- resolution  13C and 15N  cross- polarization magic angle spinning (CP/MAS) NMR spectra of three natural  melanin solids: Sepia officinalis  melanin, Sepia officinalis  melanin free acid  (MFA) and Human hair melanin . The functional group  characterization of  Human hair  melanin by  NMR  is  the  first  to date and  the 13 CCP/MAS NMR  spectra reported  here show improved  resolution  of chemically  inequivalent sites. The observed  spectral regions of the  solid melanin samples can be assigned to the postulated structural unit of the polymer chain  of  Sepia MFA derived from solution – state  NMR studies .  To  assist in the assignment of functional groups in the spectra, the solid – state CP/MAS NMR spectra are compared with high – resolution  13 C and 15 N CP/MAS spectra of four model compounds,  L-dopa, dopamine, 2-methoxycarbonyl-3-ethoxycarbonyl-4- methylpyrrole and ethyl 5,6-dimethoxyindole-2-carboxylate.  To aid further in the assignment of protonated and non- protonated  carbon atoms, CP contact time dependence  and non- quaternary carbon suppression (NQS) experiments were performed on the melanin samples.  The  15N CP/MAS spectra of the melanin samples  confirm the  presence of indole and  pyrrole units in the melanin polymer . The  NMR peaks observed in all of the melanin samples  are relatively broad, presumably owing to the presence of free radicals. Electron spin resonance  (ESR) data shows that all three melanin samples contain localized  free radicals (g=2.007), with the Sepia melanin containing a 10- fold higher free radical density than Human hair melanin.  

 

 

 

 

-----------------------------------------------------------------------------------------------------------------------

It was reported  that  in sepiomelanin dopachrome units are present (  M.Piattelli, R.A.Nicolaus  The structure of melanins and melanogenesis-I-The structure of sepiomelanin in Sepia  Tetrahedron 15, 66-75,  1961  ).

It would be interesting to compare the sepiomelanin spectrum with those of Dopa-melanin, Cyclodopa-melanin, DHI-melanin and DHICA-melanin.

Samples of melanins BCM must be composed of pure not degraded granules

###############################################################################.

      166.   Unlocking the molecular structure of fungal melanin using     13C biosynthetic labeling and solid-state NMR

               S.Tian, J.Garcia-Rivera, B.Yan, A.Casadevall, R.E.Stark

              Biochemistry 42, 8105-8109, 2003

 

Melanins  are enigmatic pigments found in all biological kingdoms that are associated with variety of  functions, including microbial virulence . Despite being ubiquitous in nature , melanin pigments have  long resisted atomic- level structural examination because of their insolubility  and amorphous organization. Cryptococcus neoformans is a human pathogen  fungus that melanizes only  when provided with exogenous substrats, thus  offering a unique system for  exploring  questions related to melanin structure at the molecular level.  We have exploited the requirement for exogenous substrate in melanin synthesis as well as the capabilities of high – resolution solid- state nuclear magnetic resonance (NMR) to establish  the predominantly  aliphatic  composition of l – dopa melanin  and to introduce 13C label  that permit the identification of proximal carbons in the developing  biopolymer. By sweep solid melanin samples in organic solvents and using two – dimensional heteronuclear  NMr conjunction with magic- angle spinning, we have identified chemical  bonding patterns  typical of alkane,  alcohol , ketone, ester, and indole functional groups. These  findings demonstrate the feasibility  of a novel approach to determining the structure of  melanin using metabolic  labeling  and  NMR spectroscopy.

 

 

 ########################################################################

167.   Cross-polarization/magic-angle-spinning nuclear magnetic resonance in selectively 13C-labeled synthetic eumelanins

 

P.Reinheimer, J.Hirschinger, P.Granger, P.Breton, A.Lagrange, P.Gilard, M.A.Lefebvre, N.Goetz

 

BBA, 1472, 240-249, 1999

 

We report a solid – state  NMR study  of synthetic eumelanins prepared by oxidation of 5,6-dihydroxyindole  (DHI) selectively 13 C-labeled at positions 2 or 3 of the indole ring. The 13C-1H couplings have been used to quantify  the carbons by selecting the non-protonated and protonated  carbon resonances. By comparing the data of non- labeled melanin to that obtained using (2-13C)-and (3-13C)-enriched DHI it was possible to clearly  demonstrate the high chemical reactivity of position 2 and, to a lesser extent, position 3 of the DHI unit.  These two sites together are responsible for three- quarters of the proton loss during  polymerization. The cross-polarization/magic- angle – spinning  spectra likewise point to a partial oxidation of positions 2 and 3 to the carboxy and carbonyl oxidation states during the formation of melanin. Furthermore, it is shown that 13C-13C dipolar interactions in (2-13C) –enriched  DHI melanins can be observed using radiofrequency – driven dipolar  recoupling (RFDR) 2D experiments .  An upper limit of about 4 A°  for the distance between the C-2 carbons is deduced  from the RFDR  experiments. This result is in agreenment with the basic arrangement of the different  atoms expected in the DHI melanins.

 

 

 

 

###############################################################################

 

 

168.       Quantitative analysis by 13C solid-state NMR of eumelanins

 

M.Herve, J.Hirschinger, P.Granger, P.Gilard, N.Goetz

J.Chim.Phys. 91,881-887, 1994

The technique of cross-polarizationCp/MAS offers many possibilities in the study of insoluble molecules such as eumelanins. A dipolar dephasing of 43-ms permitted a determination of the resonance due to quaternary carbons. Moreover after an extremely short polarization transfer time ( 35 ms ) it is possible to selectively differentiate the protonated carbons. The  spectra obtained by these two methods associated with those resulting from a cross-polarization experiment for a duration of 2-ms permitted a quantitative measurement of the ratio of protonated to non-protonated carbons in natural and synthetic melanins. The quantitative aspect of CP/MAS was verified by a comparison of MAS and CP/MAS spectra

 

 

################################################################################.

  169. Synthesis of dopamines labeled with 13C in the alpha or beta  side chain position and their application to structural studies on melanins by solid-state NMR spectroscopy

 

Liebigs Ann.Chem. 563-567, 1994

 

O.Crescenzi,C. Kroesche, W.Hoffbauer, M.Jansen, A.Napolitano, G.Prota, M.G.Peter

 

Solid state NMR spectroscopy was applied to the analysis of melanins prepared by peroxidase / H2O2     oxidation of dopamines specifically 13C labeled in the alfa or beta chain positions: A surprisingly diverse patterns of signals indicated the presence of uncyclised dopamine and noradrenaline-derived units,in addition to indole and carbonyl carbon atom.These structural features,coupled with the results obtained from elemental analysis of dopamine melanin samples prepared under different conditions, point to a pigment formation process more complex than previously believed

 

 

 

#####################################################################.

 

170. NMR studies of melanins : Characterrization of a soluble melanin free acid from Sepia ink 

S.Aime, M.Fasano, E.Terreno, C.J.Groombridge

Pigment Cell Res., 4, 216-221, 1991

This paper  deals with the nuclear magnetic resonance characterization of a soluble derivative  (melanin free acid ) of  Sepia melanin  obtained by a  peroxodative treatment  of the parent (insoluble) species.  High resolution 13C and 15N  solid state NMR  spectroscopies allow the assessment  of the chemical changes occurring  in the macromolecule upon  solubilization. 1H and 13C NMR  solution spectra are discussed in light of the  results obtained from the solid state  spectra. Furthermore, the coordination properties of melanin have been investigated through  27 AI NMR spectroscopy and proton  relaxation enhancement studies of the  paramagnetic  gadolinium complex of melanin  free acid .  T hrough these experiments it has been possible to evaluate the molecular reorientational  time  TR (and from it an estimated molecular weight close to 20 KDa) and the strength of the metal- macromolecule interaction.  

 

 

 

 

 

###############################################################################.

171. Solid –state 13C NMR characterization of melanin free acids from biosynthetic and naturals melanins

 

S.Aime, M.Fasano, C.Croombridge

 

Gazz.Chim.Ital. , 120, 663-664, 1990

13C Solid – State CPMAS  NMR  spectroscopy is used to evaluate the effect of H2 O2  on natural  and biosynthetic melanins. The 13C spectrum of a biosynthetic melanin prepared in the presence of catalase is quite different from the corresponding  13C spectrum of a natural melanin.  However. The reaction of  H2 O2  on the biosynthetic  melanin affords a water- soluble product (MFA, Melanin Free  Acid ), whose 13C spectrum  shows similarities with the  natural  pigment. In addition to provide a bridge between solid and solution state , these results may be relevant to understanding  the role of melanin in removing  H2 O2 from living  system.

 

 

 

##########################################################################.

172.     On the structure of eumelanins : Identification of constitutional patterns by solid-state NMR spectroscopy.

 

M.G.Peter, H.Forster

Angew.Chem.Int.Ed.Engl. , 28, 741-743, 1989

 

 

 

 

 

############################################################################.

173.  13C Solid-state Cross.polarization/magic-angle-spinning Nuclear Magnetic Resonance spectra  of natural and synthetic melanins

 

S.Aime, P.R.Crippa

Pigment Cell Res., 1, 355-357, 1988

 

The  availability of  NMR spectrometers operating in cross polarization/ magic angle  spinning (CP-MAS) has provided a powerful tool for the structural  elucidation of  insoluble materials . In this 13C NMR  study of eumelanins we report the first direct  evidence of the presence of different chemical functionalities in synthetic and natural  eumelanins. These spectra contain useful information for the characterization of melanins from different sources.

 

 

###################################################################.

174 .     Analysis of the structure of synthetic and natural melanins by solid-phase NMR

G.A.Duff, J.E.Roberts, N.Foster

Biochemistry, 27, 7112-7116, 1988.

The structure of one synthetic and two natural melanins are examined by solid state NMR using cross polarization,  magic  angle sample spinning,  and high- power proton decoupling. The structural  features of synthetic dopa melanin are compared to those of melanin from malignant melanoma cells  grown in culture and sepia melanin from squid ink.  Natural abundance 13 C and 15N spectra show resonances  consistent with known pyrrolic  and indolic structures within the heterogeneous biopolymer, 13Cand 15N spectra show resonances consistent with known pyrrolic and  indolic structures within the heterogeneous biopolymer; 13C spectra indicate  the presence of aliphatic residues in all three materials. These solid –phase experiments illustrate the  promise  of solid- phase NMR for elucidating structural  information from insoluble biomaterials.

 

 

 

 

 

#############################################################

175. Solid-state analysis of eumelanins biopolymers by electron spectroscopy for chemical analysis

Anal.Chem. 62, 949-956, 1990

B.Clark, J.Gardella, T.Schultz, D.Patil

 

Non scrivere

 

 

 

 

 

######################################################################.

176. A 13C solid-state NMR studt of the structure and auto-oxidation process of natural and synthetic melanins

BBA,  1204, 19-27, 1994

M.Herve, J.Hirschinger, P.Granger ,P.Gilard, A.Deflandre, N.Goetz

 

The paper  presents a 13C CP/MAS NMR  study of the melanin pigments obtained  natural and synthetic  origins: sepia-melanin from  squid ink and three synthetic  5,6-dihydroxyindole- melanins  prepared using different non-enzymatic oxidation  pathways.

The synthetic pigments can be distinguished  from natural melanin by the absence of aliphatic carbons, thereby confirming the unreacted 3.4- dihydroxyphenylalanine  and the proteinaceous origins of the  aliphatic resonances in natural  eumelanin.

The spectra of selected non-protonated carbon resonances and those with only protonated carbon signals led to a  quantitative analysis. An auto-oxidative  experiment using  asynthetic melanin, over a period of 130 h, has shown an unusually slow  disappearance of hydrogen peroxide formed in situ. The 13C-NMR spectrum of the insoluble oxidized synthetic melanin  compared to that before auto- oxidation  clearly demonstrates that the oxidation  process is  associated with chemical  changes within the pigment; i.e., carbonyl  functional group formation and an increase of the non-protonated carbons fraction.

 

 

#######################################################################.

 

 

 

UV, IR, EPR

 

 

 

 

 

 

 

 

 

 

bruno.nicolaus@virgilio.it            Biochemistry     parisi@unina.it            Biochemistry

rnicolaus@tightrope.it                 Chemistry       shah_hussain@msn.com   Computer models

Ringraziamo il personale della Biblioteca della Stazione Zoologica di Napoli per la preziosa assistenza data.

 

 

 

Naples , March, 2003

Revised  Februar 2004