Link 9 -Animal colours
With laboratory notes
www.tightrope.it/nicolaus/index.htm
There
are no blue or green pigments in Animal kingdome.
Summary.
Black materials are widespread in animals, plants, soil
(humic acids), and interstellar space. The black which we see in animal
tissues, (skin, hair, fur, eyes, scales, feathers, cuticle, etc) is due to
the presence of melanin. Melanin is produced by cells called melanocytes and
melanophores typical of cold-blooded vertebrates. In the cells the black
is synthesised in organelles, called premelanosomes and melanosomes, until
the formation formation of granules of the characteristic form and size
(PICTURE I). In the melanophores the granules of melanin can move; this movement
is under hormonal control. The movement of the granules can modify the colour of
the animal.
The black is not only black but contributes to the formation of blue, green and
yellow (PICTURE 2). The blue is seen because of the Tyndall effect and the green
because of the sum of the Tyndall blue and a yellow pigment (often a carotenoid).
The various colours are shown by the small bird the Bengal Pitt in PHOTOGRAPH 2
and the green and blue colours in the tree-frogs in PHOTOGRAPH 3. Animals can
appear black if the granules are dispersed in the melanophore and lighter if
they are concentrated.
The melanins are formed by oxydation of o.phenols and o.aminophenols (melanogens)
with the formation of polyquinones in
the hydrate form. The reaction would seem to be controlled by the
tyrosinase enzyme, irrespective of the nature of the melanogen. A
melanogen much studied is the 5,6-dihydroxyindole (DHI), but it has not be
proven that DHI-melanin is the pigment cell. The hydrated form of the
polyindolquinone (TABLE 3) is little toxic for the cells and shows a structure
in accordance with, centesimal, IR spectrum, MALDI and MALDI-TOF spectrum,
analysis.
The melanins are radical-polarons which have a base unit which is in piles in
the form of graphite sandwiches with interspacing of 3.5 Å or in fullerene
structures with interspacing of 4.4 Å. The differences in the interspacing
should be characteristic of the vegetal melanins and the animal melanins. (see
TABLE 2)
The polyindolquinone structure constitutes a bed of hydroxyls on a framework
theoretically able to assemble of cells and therefore to the construct tissues.
Recent studies have shown that the melanins are sensible to heating and to
radiation. A special break up is operated by LASER and this can be followed by
MALDI and gas-chromatography. Characteristic fragments can be also obtained also
by pyrolisis at the Curie point or atomic bombardment FAB.Melanins
are sensitive to sonication.
The melanins are amorphous semi-conductors or superconductors. They appear black
because of the small value (eV) of the gap between the valence band and the
conduction band (PICTURE 3). Because of their electrical properties the melanins
can be considered an auxilliary means of communication between tissue and the
central nervous system.
The chemical data, and in a minor part the physical data, collected so far, are
uncertain because they derive from studies carried out on heterogenous material
and artefacts : they must be revisited.
Sommario
introduttivo ed esplicativo.
Il
materiale nero è molto diffuso negli animali, piante, suolo (acidi umici),
spazi interstellari.
Il nero che noi vediamo sui i tessuti degli animali (pelle, capelli, peli,
pellicce, occhi, squame, piume, cuticole etc.) è dovuto alla presenza di
melanina. La melanina è prodotta da cellule dette melanociti e melanofori.
Nelle cellule il nero viene sintetizzato in organelli detti premelanosomi e
melanosomi fino alla formazione di granuli dalla caratteristica forma e
grandezza. (DISEGNO 1). Nei melanofori i granuli di melanina si possono muovere;
il movimento è sotto il controllo ormonale. Il movimento dei granuli può
modificare il colore degli animali.
Il nero non è solo nero ma contribuisce alla formazione del blu, del verde, del
giallo (DISEGNO 2). Il blu si vede per effetto Tyndall e il verde per effetto di
somma del Tyndall blu con un pigmento giallo (spesso un carotinoide). Le varie
colorazioni sono mostrate sul piccolo uccello Bengala Pitt della FOTO 1 e il
colore verde e blu nelle raganelle della FOTO 3. L'animale può apparire nero se
i granuli sono dispersi nel melanoforo e chiaro se concentrati.
Le melanine si formano per ossidazione di o.fenoli e o.aminofenoli (melanogeni)
con formazione di polichinoni nella forma idrata. La reazione sembrerebbe sotto
il controllo dello enzima tirosinasi a prescindere dalla natura del melanogeno.
Un melanogeno molto studiato è il 5,6-diidrossindolo (DHI).
La forma idrata del polindolchinone (TAVOLA 3) è poco tossica per la cellula e
mostra una struttura in accordo con la analisi centesimale, lo spettro IR, e con
l'analisi dello spettro MALDI e MALDI-TOF.
Le melanine sono dei radical-polaroni le cui unità di base si impilano a
formare sandwich grafitici con interspazi di 3.5 A° o strutture fullereniche
con interspazi di 4.4 A°. Le diversità degli interpazi sarebbero
caratteristiche delle melanine vegetali e di quelle animali.
La struttura polindolchinonica nella forma idrata costituisce un letto di
ossidrili su di una impalcatura abile teoricamente allo assemblaggio cellulare e
quindi alla costruzione di tessuti.
Da recenti studi risulta che le melanine sono sensibili al riscaldamento e alle
radiazioni.Una peculiare frantumazione viene operata dal LASER e che può essere
seguita col MALDI e la gas-cromatografia. Caratteristici frammenti possono
essere ottenuti anche per pirolisi al punto Curie o dal bombardamento atomico
FAB.
Le melanine sono dei semicoduttori o superconduttori amorfi. Esse appaiono nere
per il piccolo valore (eV) del gap fra la banda di valenza e quella di
conduzione (DISEGNO 3). Per le loro proprietà elettriche le melanine possono
essere considerate un mezzo di comunicazione ausiliare fra tessuto e tessuto e
fra tessuto ed SNC.
I dati chimici e in minore parte quelli fisici raccolti fino ad oggi sono
incerti perche derivati da studi eseguiti su materiali eterogenei ed artefatti :
essi debbono essere rivisti.
PART I
Papers
to be read
W.Chavin,
Fundamental Aspects of Morphological Melanin Color Changes in Vertrebate
skin, Am.Zoologist, 9, 505, (1969).
Although
integumental tyrosinase activity is usually correlated with the degree of
melanoderma, exceptional cases are sufficiently documented to provide a basis
for the presence of normal melanogenic control mechanism in the skin. In
addition, the enzyme may not always be bound to a subcellular organelle, thus
suggesting its origin is at a distance from its site of action (premelanosome).A
number of biological factors affect the enzymatic activity and its subcellular
distribution indicating that the biological state of the organism cannot be
disregarded in biochemical studies. Further,the use of variously labelled
substrates has revealed the pokilopolymeric nature of melanin and the
possibility of the direct effect of the intracellular environment upon the
nature of the polymer. Several types of primary control mechanism directly
affecting the activity of tyrosinase are present in the vertebrate integument.
It is probable that additional mechanisms will be uncovered, eventually.
C.L.Ralph
''The Control of color in birds '' American Zoologist 9, 521, (1969).
The control of birds result from deposition of pigments, mainly melanins and
carotenoids, in integumentary structures chiefly the feathers. The plumages of
birds indicate their age, sex, and mode of living, and play important roles in
camouflage, mating, and establishment of territoires. Since feathers are dead
structures, change of color of feathers is effected through divestment (molt)
and replacement The color and pattern of a feather are determined by the
interplay of genetic and hormonal influences prevailing in its base during
regeneration. Most birds replace thei feathers at least once annually.Some wear
the same kind of basic plumage all the time but others alternate a basic and
breeding plumage, either in one (the male) or both sexes. Stil others may have
more than two olts, adding supplemental plumage at certain times in the plumage
cycle. The varieties of patterns of molt, the kinds of plumage, and the colors
and patterns of eathers among birds apparently are the result of several kinds
of selection pressures working through evolution
In Anolis the ability to
adapt to a background is dependent upon the level of circulating MSH, the
release of which is dependent on information received through the eyes. Blinded
lizards are brown under conditions of strong illumination and green under
conditions of lower light intensities, and,again, these color changes are
regulated by MSH. Color changes in the blinded lizards are regulated by dermal
photoreceptors. High or low temperatures directly affect the color of Anolis
skins and alter the rate at which skins responds to hormones. Aggregation of
melanin granules within Anolis melanophores in response to
sympathomimetic stimulation is regulated through alfa-adrenergenic receptors
whereas dispersion of melanin granules in response to such stimulation is
controlled through beta-adrenergic receptors possessed by the melanophores. Most
Anolis melanophores possess both alfa and beta adrenergic receptors, but
some melanophores possess only beta adrenergic receptors. In the normal
physiology of the lizard, under conditions of stress, stimulation of alfa
adrenergic receptors by catecholamines leads to an '' excitement-pallor ''
followed by an '' excitement-darkening '' resulting from stimulation of beta
adrenergic receptors which causes dispersion of melanin granules within
localized populations of melanophores. Thus, in Anolis, dispersion of
melanin granules within melanophores is regulated by both MSH and by
catecholamines. Evidence is acquired that the intracellular level of cyclic AMP
within melanophores may be responsible for the regulation of movement of melanin
granules.
E.Florey
Ultrastrutucture and Function of Cephalopod Chromatophores from
Department of Zoology, University of Washington, Seattle, Washington 98105 (9) :Each
chromatophore organ consists of a pigment cell and of several radial muscles
fibers that represent separate cells. The pigment granules are contained within
an elastic sacculus within the pigment cell. The sacculus is attached around the
equator of the chromatophore to the cell membrane by zonal haptasomes. In turn
the cell membrane is attached to the radial muscle fibers by a dense basal
lamina. The cell membrane of the retracted chromatophore is highly folded.
Contraction of the radial muscle fibers is initiated by an excitatory junction
potentials,byminiatures potentials, or by spike potentials. The latter arise
spontaneously in the muscle fibers when these have undergone some internal
change. The contraction of the muscle fibers causes expansion of the
pigment-containing sacculus. Relaxation of the muscle fibers permits the
sacculus to assume its original lenticular or near -spherical shape; the energy
for this is stored within the expanded elastic components of the sacculus. In
normal skin the chromatophore organs are entirely under the control of the
central nervous system, the muscle fibers being activated only by local,
excitatory postsynaptic potentials initiated by motor nerve impulses.That
postsynaptic potentials are nonpropagating insures that individual motor fibers
can be activated individually, thus permitting a delicate control of the skin
color by recruitment as well as by frequency. Tonic contractions and
pulsations,involving spontaneous release or trasmitter from nerve terminals and
spike generation within the muscle fibers,respectively, are the result of
altered, abnormal conditions within the skin.
---------------------------------------------------
The green colour of dragonflies is produced as showed
in drawing 2 . In all cases there
is a layer of dark pigment behind the scattering cells. In the
green hairstreak butterly,Callophrys rubi, the green underside of
the wings in a interference colour and in the green forester moth, Procris
statices, the colour is also structural. In the large moth Urania
rhiphoeus of Madagascar structural green is seen. In green caterpillars and
orthopterans the colour of the integument, and often also of the blood, is due
to a mixture of a blue with a yellow pigment; the blue is a biline and the
yellow a carotenoids. In the green phase of the prawn Hippolyte varians
a yellow carotenoid and a blue carotenoprotein are juxtaposed. The green gills
of some cultured oyster are coloured by a bluish pigment, from diatoms, together
with a yellowish pigment in the gills. The greenish sponge Halichondria
panicea has a yellow carotenoid together with a blue pigment. Olive-green
coloration in the feathers of some birds, in the fur of the green monkey and on
the wings of some butterflies is caused by the apposition of black and yellow
elements. Crustaceans have a green pigment, a caroteneprotein, familiar in the
green shore crab. A similar pigment is ovoverdin of lobster eggs and the green
pigment of Daphnia eggs. The starfishes Marthasterias glacialis
and Asterina gibbosa, and a variety of the breadlet anemone, Actinia
equina, are also coloured by green carotenoproteins. The green colour of
chlorocruorin, seen in blood vessels of sabellids and serpulids, results in body
coloration only in chlorhaemid worms. Another important green pigment is
biliverdin. It is seen in the bile of amphibians, birds and some mammals, in the
egg shells of some birds, in the bones of some fishes, in the roots of some
rhizocephalan parasites and in the base of the beadlet anemone. Other green
tetrapyrrole pigments, related this time to chlorophyll, are bonellin which
colours the integument of Bonellia viridis and phaeophorbides in the
gut-wall of the polychaetes Owenia fusiformis and Chaetopterus
variopedatus. Haemovanidin, found in ascidians, is a third green blood
pigment.
There are several green pigments of unknown chemical nature. The frog has green
rods in its retina ; turacoverdin is found in the feathers of some touracos;
there are green pigments in certain moths; a green pigment colouring the bug Psylla
mali on apple trees is apparently formed by simbiotic bacteria; a green
pigment has been found in the integument of the lugworm, Arenicola; there
are green schemochrome ( physical colour ) which colour the polychaetes Eulalia
viridis and Phyllodoce viridis, and there is a dark-green
schemochrome in the entomostracans Triops
and Cypris.
From Teresa Strzelecka,
Physiol.Chem.Phys., 14, 219-231, (1982).
DOPA-melain dependence of dark
current on temperature in the range 298-333 K was measured as well as dependence
of optical absorption coefficient on wavelength in the range 250-800 nm. It was
found that up to 311° K thermal activation energy equals 0.1 eV and above 313°
K it equals 0.78 eV. The first value is connected with the band of localized
states at the Fermi level.Optical gap, determined from optical absorption
measurements is equal to 1.40 eV. The estimated value of so, assuming
the value of thermal coefficient of activation energy to be g =5 x10-4
eV / K°, is 2 x 10-6 W-1 cm-1 for 0.1 eV and 5 x 102
W -1 cm-1 for 0.78 eV. Density of states in the valence
band is N (Ev) = 8 x 10 21 / cm 3. eV and in
the band of localized states at the Fermi level N (Ef) = 3 x 10
13 / cm 3. eV.
Basic semiconductor characteristics of natural melanins isolated from bovine
eye, human dark hair, and banana peel were obtained by means of the dc
dark conductivity experiments and optical absorption measurements. The results
were compared with results obtainedfor synthetic melanin.
Specific conductivity in natural melanins is of the order 10 -11 W -1
cm -1 and in synthetic melanin 10 -8 W-1 cm -1.
Thermal activation energies in the range 298-333° K are eye melanin 0.93 eV ;
hair melanin, 1.01 eV; banana melanin, 1.04 eV; whereas synthetic melanin has
two values of activation energy : up to 311° K, 0.1 eV ; above 313° K, 0.78 eV.
Optical gaps are : in eye melanin, 1.73 eV; in hair melanin, 1.35 eV; in
bananamelanin1.55eV; and in synthetic melanin, 1.40 eV.
-------------------------------------------------------------
Conducibility
may change with
:
1. Stable open-shell (free
radical).
2. Band overlap, small HOMO-LUMO, large bandwidth
3. Molecules with delocalized p -molecular orbitals
4. Inhomogeneous charge and spin distribution
5. Segregated stacks or sheets of radical species
6. No periodic distortion which opens a gap in the
density of states across the entire Fermi surface
7. Little or no disorder
8. Molecular components of appropriate or compatible
size
9. Fractional charge tranfer or mixed-valence material
10. Strong interchain coupling or sheets to suppress metal to
insulator(M-I) phase transitions
11. Cation/anion nominally divalent
12. Polarizable species to help reduce U.
---------------------------------------------------
A
characteristic dimension of a melanin protomolecule synthesized from tyrosine
has been investigating by scanning tunneling microscopy (STM). Identification of
a melanin protomolecule of approximately 20 Å lateral extent and circa 10 Å
height has been established. This size is in good agreement with models
constructed to fit wide angle X-ray diffraction experiments on melanin. These
protomolecules are believed to consist of Van der Waals interacting stacks of a
basic random polymer of 5,6-indolequinone units (in forma idrata ndr.). There is
extensive p -delocalization within the individual polymeric sheets. Structure
minimization and molecular orbital tecniques were employed to verify the X-ray
and STM results (10).
---------------------------------------------------
From Maria Grazia Bridelli, Biophysical Chemistry, 73,
227, (1998).
The problem of characterizing the heterogeneous
melanins was approached by means of light scattering techniques, static and
dinamic. The static technique allowed us to identifythemacromolecular properties
[ (MW) and (R2g)1/2 ] of melanin extracted from
sepia ink sac and two synthetic analogues : L-DOPA -melanin obtained by
autoxidation and by enzymatic oxidation by Tyrosinase.
By dinamic light scattering (DLS), the hydrodynamic radius Rh was
measured to monitor the temporal behaviour of the polymerization and aggregation
processes and Rh variation by changing the chemical constraints of
the polymerization medium, such as pH and ionic strength. The fractal dimension
of the aggregates of melanin, both natural and synthetic, in the past 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 that are spherical not largely hydrated and
self-assembled, following a reaction limited aggregation kinetcs (d = 2.38).
--------------------------------------------------------------------------
PYRROLE RING IN MELANINS
From Z.E.Jolles, Chemistry and Industry, pag.846,
August 8, (1953).
The
importance of pyrrole compounds in the formation of melanines (hair, skin, eyes,
tumors,etc.) was first pointed out by Angeli. In 1915 he put forward the
hypothesis that such pigments were produced as the last stage of an enzymatic
oxidation and polymerization of pyrrole derivatives originated from proteins or
produced from phenolic amino-acids such as tyrosine and congeners. He surmised
in 1918 that an oxidative fission of the benzene ring, followed or preceded by
the pyrrole ring formation, must be involved in thr biogenesis of melanines.
Although a partial oxidation of benzene (to muconic acid) in the animal body, in
most cases occurring preferentially in the aliphatic chain if present was
already known the chopping out of a benzene ring in order to account for the
striking similarity of the pyrrole-black with natural melanines, was undoubtedly
a daring and highly speculative hypothesis. It formed the basis of Angeli's
subsequent investigations with his co-workers in this field (1915-1930), but
also earned him authoritative rebukes.
The discovery of 5,6-dihydroxyindole in 1926 by S.H.Raper, in the initial stages
of the production of melanine from tyrosine by tyrosinase from the meal worm,
while successfully realizing one anticipation of the above biogenetic theory of
melanine formation, i.e. via an indole derivative, made the second anticipation,
viz. that of an oxidative rupture of the benzene ring between the two hydroxy-groups
leading to a pyrrol compound, a feasible proposition.
Unfortunately, except for the instability of the few described pyrrole acetic
acids which readily decarboxylate, little is known to indicate their possible
metabolic paths or to warrant speculation. It is surprising how little weight,
apart from the Italian workers has been given in the experimental field, to the
study of pyrrole compounds in relation to melanogenesis, although the
alternative speculation, viz. concerned exclusively with the possible
arrangements of the whole indole unit of Raper's melanine precursor, in the
polymeric end-product, have yielded useful information. The recently reported
ring opening between the two hydroxygroups of catechol and, presumably at the
corresponding positions, of 5,6-dihydroxyindole, with evolution of carbon
dioxide, by simply bubbling air through the aqueous alkaline solutions, is of
considerable interest in this connexion. An earlier observation that the uptake
of oxygen during the enzymatic oxidation tyrosinase (9), in the presence of air,
of cathecols to o.quinones, is much greater than that required for the
production of the latter and the conclusion that other substances of unknown
constitution are produced either by spontaneous change or further oxidation, are
highly relevant.
Of no less significance is the recent development of the Woodward hypothesis of
biogenesis of strychnine applied by Robinson to the case of emetine, and by
Prelog to cinchonamine and cinchonine, based on the oxidative fission of the
benzene ring between two orthohydroxy groups, followed by a transformation and
partial recombination of the fragments thus produced, in a different manner.
In the light of the above development, Angeli 's theory of melanine formation
and the working hypotheses which with a number of variants it still provides,
deserves attention; its reconsideration appears to be justified both by the
degree of coincidences which recall for it, and by the results from independent
speculations in other fields, in particular those based on biogenetic changes of
molecules having in common the fundamental indole or catechol group.
Melanins from interstellar spaces
See the papers ( link 12 ,link 13, link 14,link 15 ,link 16 )
Between the Southern Cross and the rich Carina region, on the southern border of
Centaurus,is a large, almost featurless emission nebula, IC 2944. It is against
this uniform, bright, backdrop that we see a small group of dark clouds of the
kind known as Bok globules. They are named for the Dutch-American astronomer who
first drew attention to them as possible sites of star formation. These dark
markings are discrete, opaque dust clouds, the largest containing enough
material to form several stars the mass of the sun. The globules are not some
line of sight coincidence ; the brightened rim of the largest clearly shows it
to be associated with the nebulosity of IC 2944.
(From
D.Malin, Anglo-Australian Observatory).
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PART
II
Melanin is clearly visible in animals and is present in the skin, hair, fur,
choroids, eyes, scales, feathers, and cuticle(1) (2). Melanin is also found in
non-visible anatomic parts like the substantia nigra of the human brain
and the locus coeruleus and the liver, and it accumulates in pathological
forms in the so-called melanoma. Melanin is an insoluble heterogeneous black
material which is difficult to purify and characterise by organic chemistry
methods, but it can be purified and characterised with the methods of
nanochemistry and especially with that of solid state nanophysics. Some animal
melanins are polyindolquinones (derived from DOPA) in the hydrate form,
characterised by a radical-polaron system also called a spine, (TAVOLA 2 e 3).
The melanins are solid materials which behave as amorphous semiconductors. The
colour originates from the transitions in materials with band structure and is
different(black,red,yellow) according to the width of the prohibited band (colour
bands) (DISEGNO 3). Chemical factors, as for example the cysteine, intervening
in the process of melanogenesis produce colourations of brown, red and yellow
with different gradations and hues. While the fur of the black guinea pig is
formed by 3,4-dioxy-phenylalanine (DOPA) the reddishness which can be seen in
the red guinea pig of PHOTO 1 is formed from cysteinildopa. The colours fawn,
orange and yellow are attributed to simple pheomelanins, with various
denominations which have changed with the advances in research, like
tricosiderin, or Cl, C, B, E, and F composites, or pyrrotricoles, or
tricochromes etc..(2), (2e), (2h), which are easily extractable and
crystalisable, while other less soluble and crystalisable substances, coloured
brown and red-brown, are to be attributed to the more complex pheomelanins.
In the bison Bison bison the coat of the newborn appears reddish because
of the lightening action of the cysteine, while the adult is reddish black.
Among mammals the kangaroos form an almost uninterrupted series of living
examples, possessing coats of brown, reddish-brown, and flame-red of tryptophan
origin. Different metabolites of the tryptophan like the acid 3-oxyanthranilic,
kinurenine, cinnabaric acid indicative of a tryptophan metabolism (3) or the
hide of the kangaroos Megaleia rufa and Trichosurus vulpicola.
The melanins are usually divided into eumelanins, pheomelanins and allomelanins
(2) which originate from uncoloured precursors called melanogens with differing
chemical structures. The noted melanogens of the animal kingdom are o.diphenols,
derived from phenylalanine and from tryptophan. The eumelanins which form from
indolic precursors are typical of mammals, the pheomelanins of a lighter colour
originate because of the intervention of cysteine in the process of
melanogenesis (hair, fur, feathers), and the allomelanins which originate from
nitrogen-free precursors are typical of plants and of microorganisms. In effect
the reactivity of the intermediates with the substances occasionally present
determines that this chemical classification is often not exact. Besides it must
be noted that the small number of pigments examined does not allow a
statistically satisfactory manner of knowing the origins of melanins and their
structures.
Melanin which appears in the form of granules is produced by cells called
melanocytes or melanophores. Apart from morphological differences the diversity
of the two cell types seems to lie in the fact that in the melanophores the
granules of melanin can move, contributing to the change in colour of the
animal, while this does not happen in the melanocytes. In certain cases the
concentration or the dilution of the granules can bring about modifications of
colouration.
the black granules allow vision of the Tyndall blue colour or of green because
of the effect of the Tyndall blue and a yellow pigment. The granules can also
contribute to modifications in colour because of reflection, diffraction and
diffusion effects of light. All the colours reported in this text are structural
colours (schemochromes or physical) and do not correspond to chemical formulae.
These colours are clearly visible in the small bird the Bengala Pitt in PHOTO 2,
where both biochromes and schemochromes are present.
Little is known about the function of melanin if one excepts its protective
action for the skin of man or mimetic function in animals. The significance of
melanin in the ear, in the eye, in the nervous system and in melanomas is not
clear. Pathological colours, sometimes induced by mutations, are albinism which
comes about through the loss of melanin, and blue frogs with the loss of the
yellow pigment PHOTO 3. Pheomelanins can be produced in the skin and in hair of
black people through the action of radiation with a high energy content; the
radiation can also modify the colour of the eyes in a non-permanent way.
The melanins are polyquinones in a hydrated form characterised by a radical-polaronic
system with stable unpaired electrons. The polymeric units are made up of 12-16
monomers, according to type of reaction used, they can be settled in graphite
sandwiches (interspacing 3.4 A°) or in fullerene cages (interspacing 4.4 A°).
The melanins are natural amorphous semiconductors with a model which corresponds
qualitatively to that of bands in semiconductors and superconductors. The
electrical conductivity, the small gap, the colour, and the EPR signal of these
materials are in agreement with this definition. Size, form and properties of
the black particles of synthesis depend on various environmental factors like
temperature, pH, concentration and doping substances.
The discoveries of some physical and chemical properties of the melanins and the
black materials, like electroactivity, superconductivity, break-up due to
radiation, communication between tissues, the capacity to organise the cells,
and the capacity to transport metals, water and gases, make these biological
materials much more interesting than could be thought. The subject is still
pervaded by a sort of interdiscplinary ignorance which makes it more and more
difficult to understand the role played in Nature by the melanins. Other
difficulties lie in the fact that the chemical and physical studies are made on
heterogenous and artificial material.
The conclusions drawn by these studies must be accepted with caution. For the
study of biological blacks, the melanins, one recommends the use of material
homogenous in the form and in the size of the granules.
The production and distribution of melanins in animals varies greatly between
species and is realised through different mechanisms in the vertebrates and the
invertebrates.
In the vertebrates the melanin is produced by the melanocytes, derived from the
neural crest in the homeotherms, and the melanophores in the pelicotherms.
Specialised organelle cells, the melanosomes,synthesise granules of melanin.
Melanin is synthesised in the various organelles starting from the various
precursors among which the best known is the 5,6-dihydroxyindols or DHI.
In the various organelles the different metabolic activities connected to the
synthesis of the melanin develop. The organelles have differing chemical
compositions (4). The organelles can be distinguished from one another thanks to
their density DISEGNO 1. In certain cases it is possible to recognise the
intermediates of melanogenesis (5) with the auxilliary of mass spectrometry (5),
(13). Melanophores and melanocytes are not clearly distinct from one another.
In the case of the sepia, one of the most studied animals, the melanosomes are
found both on the skin and in the ink sack. As is known, the animal defends
itself, if disturbed, by emitting a black cloud containing melanosomes a various
stages of maturation and with various melanogenic activities.
The deep sea sepia Heteroteuthis dispar secretes a substance from a gland
placed near the mouth which, on contact with sea water, produces a luminous
cloud (6)(7). In this case one does not speak of melanosomes, but of photophores.
All men independently of race have about the same number of melanocytes. The
different colour of the skin is to be attributed simply to a major quantity of
melanin produced by the melanocytes. The quantity of the melanocytes varies from
tissue to tissue and from the exposure of the tissue to the sun. The differences
in skin colour are due to a difference in the functional factors not to anatomic
factors. The number of melanocytes decreases with age. Besides diminishing their
activity the cells have a shorter life cycle. The form and the size of the
melanocytes varies with the density of melanocyte population.
In the epidermis the granules are exported by the melanocytes to the adjacent
keratinocytes or to the bulbous cells of the piliferous follicles, which
distribute the granules according to a variety of modalities to give the various
colours to the skin, the hair or the fur. The melanophores of warm blooded
vertebrates withhold their granules and are able to rapidly move the pigment in
response to environmental and physiological stimuli.
The invertebrates utilize a quite different mecchanism of melanine pigmentation.
During the formation of the cuticle, the epidermic cells secrete the melaninic
precursors (e.g. dopa, dopamine, catechol etc) in the extracellular matrix where
they oxidise self-catalytically or enzymatically (DHI polymerase ?) to melanin
while bonds are formed with certain proteins in the cuticle as well as with the
chitin. In insects, two other catecholamines, the N-(beta-alanildopamine) (NBAD)
[ cellulose-like biopolymers predominantly of unbranched chains of N-acetyl-D-glucosamine
residues ] and the N-acetildopamine are secreted in the same way to form the
sclerotin [ a tegumental protein ] during the formation of the non-pigmented
cuticle. The term melanophore is vague, in that the melanin can originate from
different precursors. It would be better to use the precursor name before the
term melanophore, e.g. DHI-melanophore, pterin-melanophore,
porphyrin-melanophore etc.
The vertebrates can have different colourations and skin patterns, which depend
on physical phenomena (iridescence or metallic colours of some fish and
reptiles, of the feathers of birds) or on pigments. These are contained in the
epidermis or in particular cells of the dermis, the chromatophores, which can be
black or brown coloured because of the presence of melanin (melanophore), or
yellow or red because of the presence of lipochromes (lipophores or xanthophores),
or iridescent because of the presence of guanine (guaninophore or iridocytes).
Some cell classes contain pigment granules which are clearly visible in the
microscope because of their natural colouration.
In man, the deep layers of the epidermis and dermis, the epithelial pigment of
the retina and the iris and some zones of the central nervous system contain
melanocytes, which are provided with a large number of dark brown or black
particles. The melanocyte (4) of melanoma B-16, or Harding Passey melanoma has a
particulary morphology characterised by the presence of organelles in various
stages of maturation, called premelanosomes, of melanosomes and of granules
(site of only melanin without enzymatic activity), DISEGNO 1.
In extraordinary melanogenetic organisation has been observed in the ink sack of
the sepia Sepia officinalis where an enzymatic complex works both at the
level of the dopachrome (8) (dopachromotautomerases which govern the
transformation of the dopachrome to DHI) and at the level of DHI (DHI-polymerase
in part identifiable with peroxydase).Precursors and melanogenesis are showed in
TAVOLA 1 e TABELLA 2. The melanocytes do not suffer keratinisation and do not
derive from epiblasts, but derive from neural crests of the embryo and migrate,
between the third and sixth month of endouterine life, to the dermis and thus
they penetrate the epidermis.
The melanocytes are located in the basal layer and in the spiney layer of the
epidermis. This layer has characteristic cellular elements, furnished with
branching extensions which extend a long distance towards the surface, creeping
into the interstices between the cells of the malpigian layer. They are not
linked by desmosomes and are without tonofilaments and granules of chetoialine.
The best method for identifying the melanocytes is by incubating the skin in a
solution containing the dihydroxyphenylalanine (DOPA) precursor of the melanin;
the melanocytes, which contain the tyrosinase enzyme necessary for the formation
of the melanin, convert the DOPA into melanin, colouring it black. The malpigian
cells which contain the melanosomes but not the tyrosinase enzyme remain
colourless. A more recent and sophisticated method consists in examining
cellular melanogenesis using mass spectrometry (5) (13).
The melanocytes possess a specific constituent, the melanosomes containing
melanin, large quantities of endoplasmatic or granular reticules and a large
Golgi complex. The melanosomes form in the Golgi complex as vescicles limited to
the membrane; successively they take on the aspect of eliptical organelles of
about 0.7 x 0.3 micron, wrapped in a unitary membrane and with a characteristic
internal organisation in lamella. The lamellas are often deposited in concentric
layers. In the more advanced phase of development the internal lamella structure
tends to become obscured by the accumulation of melanin and of proteins. Once
their transformation is completed, the melanosomes migrate into the extensions
and are transferred to the cells of the malpigian layer by a sort of secretion
called cytocrine.
Therefore, the melanin granules are also present in the cells of the spiney
layer but only the melanocytes, which contain the tyrosinase enzyme, synthetise
melanin.
The combination of a melanin with the adjacent epidermic cells is denominated
epidermic melanin unit. The melanocytes are always present in the basal layers
but some dendritic and clear cells are often found in the more superficial
layers. These cells, denominated Langerhans cells have obscure origins and
functions; some authors consider them to be exhausted melanocytes, but this
hypothesis is contradicted by the numerous recent experimental data which lead
to the conclusion that they are active cells.
The epidermic melanin is responsible for the pigmentation of the skin and
carries out an important role in the protection of the organism from
ultra-violet radiation. The phenomenon of suntanning is due to the activation
(?) of the tyrosinase enzyme which determines an increase in the quantity of
melanin produced and an increase in the number of the melanosomes which are
transferred to the epidermic cells. The racial differences in skin pigmentation
also depend on the level of melanisation of the melanosomes and on the intensity
of the process of transfering the granules into the epidermic cells, rather than
on the number of melanocytes present.
The pigment of hair, like that of the epidermis is essentially thanks to their
melanin content. The melanin of hair is formed by the melanocytes, which are
distributed in the upper part of the piliferous follicle bulbs. The melanocytes
in this site, like those in the epidermis, move upwards, they emit cytoplasmatic
processes which reach the epithelial cells and furnish them with melanin. These
cells meeting with keratinisation transform into the corticals and the marrow of
the hair. The melanin which they contain is incorporated into the keratin of
the hair giving it its colour.
When the cells which form following the cellular division of the follicular
matrix move upwards, they transport the melanin to the upper part of the bulb;
then, moving further and becoming keratinised they tranform in the cortical and
into the marrow of the hair. The melanin which they contain is incorporated into
the keratin of the hair giving it its colour.
In the integuments containing melanin the change of colour is due to the
quantity of melanin contained in the cells and the number of pigmented cells per
unit area of the integument. For this reason the total melanin content can be an
important factor for evaluating changes in colour. The measure of the level of
enzymatic activity, that is, the dosage of the tyrosinase can in turn be used
for the dosage of the melanin. The method of dosage most followed is that of
radiometry using the tyrosinase marked with 14C.
Apparently hair has different colours, but under the microscope only three
colours can be recognised, these being, black, brown and yellow. The yellow
pigment, called pheomelanin, and its formation seem to be under the control of
different genes to those which regulate the formation of the black and brown
melanins.
In many cases the colouration of animals are those of the semiconductors, that
is, black, red and yellow with all the various nuances due to the width of the
prohibited band (gap).
In the skin of the inferior vertebrates (fish, amphibians, reptiles), the
pigmental cells are denominated chromophores and are classified into various
categories according to the colour : iridophores, containing paracrystalline
purine, guanine, adenine and hypoxantine; xanthophores or eritrophores which
contain pterins and carotenoids; melanophores which produce dark and brown
granules containing melanin. The melanophores are distinguished from the
melanocytes by their capacity to expand and to retract their lengthening and by
their property of varying the state of aggregation of the pigment particles,
thereby determining the variations in the tint of the skin; this phenomenon is
especially noted in the skin of the chameleon and cephalopods (9).
In fish and in amphibians the iridophores can contract under the action of drugs
or of intermedin (beta MSH) and the action of this can be reversed by other
agents. The melatonin has no effect on the iridophores while the xanthophores of
some fish are expanded by intermedin. The integrated response of the
chromatophores of amphibian skin to intermedin has been described as the
mecchanism at the base of colour changes (9).
In amphibians the dispersion of the pigment is therefore under the influence of
the hormone MSH (melanocyte stimulating hormone). The mechanism of dispersion of
the pigment and therefore the colour of the animal is not yet completely clear
but it seems that the action of MSH may be considered mediated by AMP (adenosine
3',5'-monophosphate). Besides, there are experimental proofs that the dispersive
effect of the catecholamines on the melanophores of the Xenophonis laevis
is mediated by the beta-adrenergic receptors. On the other hand the effect of
the catecholamines of the melanophores of amphibians seems to be mediated the
the alpha-andregenic receptors. There is also the possibility that the effects
of the catecholamines are also through the control of the level of the cyclic
AMP in the melanophores with the stimulation of the beta-adregenics which
produce an increase in the AMP followed by the dispersion of the melanin and on
the other hand a stimulation of the alpha-adregenic which produces a decrease in
the level of cyclic AMP followed by the aggregation of melanin.The physiological
changes of colour in reptiles has been studied in depth for the Anolis
carolinensis (9).
The melanophores of the very black deep sea fish Melanocetus and Astronesthes
which have obscurable photophores are very special; the colouration of the deep
sea fish and their capacity to emit light represents a real puzzlefor science.
The skin of most of the amphibians comprises a quite deep layer, rich in
melanophores in stellated form which contain melanin in their cytoplasm. These
cells allow the skin of the amphibians to become darker and lighter. In fact, if
the granules of the pigment are dispersed in the peripheric stellate expansions
of the melanophores the animal takes on a dark tint; instead, if the pigment
retreats, concentrating at the centre of the cell around the nucleus most parts
of the surface of the body remain without pigment and the animal takes on a
light tint. This mechanism can also lead to pigments of different tonalities to
the green and blue schemochromes.
The
rapid changes in skin colour in the cephalopods has always fascinated
naturalists from antiquity, and a chromaphore organ was first described in 1819
in the work entitled '' Descrizione di un particolare sistema di organi
cromoforo espansivo- dermoideo e dei fenomeni che esso produce, scoperto nei
molluschi cefalopodi '' by G. Sangiovanni, Giornale Enciclopedico di Napoli 9,
13, (1819).
The dispersion relative the pigment in the melanophores
is under the influence of the melanophore stimulating hormone (MSH) of the
hypophysis and the response of the melanophores to changes in the production
rate of this hormone are rapid and intense (1) (9).
Besides, in the skin of amphibians, especially in the melanophore layer, there
is another layer of cells with an iridescent aspect, containing guanine and
called guanophores or iridocytes; the association of the melanophores and the
guanophores gives the skin of amphibians a colour tending to blue, able to
become darker and lighter according to the variations in the melanophores
described. Often enough an even deeper layer of lipophores, cells with a colour
which goes from yellow to red because of the presence of lipochromes, changing
with the tints of the overlying layers, gives the animal a decidedly green
colour, or, if the lipophores are abundant, yellow, orange or red. In some
species, the lipophores, like the melanophores, are also able to suffer changes
on the level of dispersion of the pigment; thus the variation in the field of
the colours of the animal skin become spectacular.
Insects are rich in melanin, among which is the cuticle. The conductor pigment
can also play a role in the visceral nervous system which controls the
alimentary canal, the heart, the excretory organs and the genitals. Examples are
the plum-sawfly Hoplocampa minuta, the setiridi Hipparechia semele,
Brintesia circe, Agapetes galathea, and the multicoloured Nifalidi.
To understand the meaning of the colour of the skin, one must consider that the
tint, in animals, besides giving the advantage of a possible camouflage against
predators in the natural living environment also allows recognition in
encounters between the two sexes and finally has the function of protecting the
underlying tissue from damage by solar radiation.
Ultimately, the movement of pigment in the chromatophores of the cephalopods,
crustaceon decapods, fish and cameleons determines changes in colour of the
animal. Considering the changes in colour of the crustaceons, the concentrating
action of the pigment is under the influence of a hormone which stimulates a
pump which exchanges sodium ions from the inside of the chromatophore with
potassium ions from the outside while the hormone which disperses the pigment
stimulates the entry of calcium ions into the chromatophores (9).
We shall come to the new, recently discovered, chemical-physical properties of
the pigment and their biological role, later.
In 1895 it was observed that an enzyme called tyrosinase, present in the
poisonous mushroom Russula nigricans was capable of transforming tyrosine
into melanin. In effect it is not to be excluded that the oxydation of the
phenols can procede through the intervention of other enzymes like laccases and
peroxydases (48). Successive studies, thanks mainly to the physiologist H.S.
Raper (1), allowed the isolation of a series of intermediaries between tyrosine
and melanin among which the dopachrome, 5,6-dihydroxyindole (DHI) the
5,6-dihydroxyindole-2-carboxylic acid (DHICA). See TAVOLA 1.
It was also observed that the melanogenesis proceeded with the consumption of
oxygen, development of CO2, and formation of H2O2.
The result was that the melanin was a product of the oxydative polymerisation of
DHI.Analysis of DHI-melanin (36),Tyrosine-melanin (49), Sepiomelanin (33), show
that the melanins are polyindolequinones in the hydrate form.
The melanogenic property of the DHICA was studied later (1e).
Recent studies (8) have established that the synthesis of black of the sepia is
controlled by different enzymes among which an enzyme which transforms the
dopachrome into 5,6-dihydroxyindole (DHI). The transformation of the DHI into
melanin is a process which can be accelerated by light, oxygen, metals and
temperature. It is not yet clear if melanogenesis can proceed with the
intervention of different melanogens like DHICA acid, and the nature of the
process in animals of different classes and orders, or the nature of
melanogenesis in differing biological sources and whether there is a difference
between physiological and pathological melanogenesis.
The oxydation of the DHI generates the 5,6-indolquinone (IQ) hydrate which on
polymerisation produces black particles which can take on their characteristic
form and size.
The macromolecular films pile up as graphite sandwiches or settle in closed
forms like the giant fullerenes (10), (11), (12), distinguishable by the values
of the interspacing in angstroms.
The PIQ polymer is generally in the hydrate form which is less toxic than the
quinone. The study of melanogenesis or better the special process which happens
in the ink sack of the Sepia officinalis is, in general, also
considered valid for what happens in the melanocytes of mammals but studies on
it are few. Effectively the tendency to copolymerisation in the various
intermediates of melanogenesis and the ability to bond organic and inorganic
products makes the process complex and little reproducible outside the cell. The
exposure to light, oxygen, to pH and to metals, and the incorrect use of enzymes
have produced confusionary elements in the study of the process of in vitro
melanogenesis.
Recently (5) it has been observed that the in vivo melanogenesis in Sepia
officinalis occurs for groups of oligomers of DHI in a very similar way to
what happens in the laboratory in the case of synthesis of melanin from DHI and
dopamine (13).
The hydrated quinonic forms appear in the course of the melanogenesis from the
first oligomers of DHI where with mass spectrometry it is possible to show the
increase of mass of 16 m/z (606, 753, 900, 1047 m/z). MALDI mass spectrometry
has recently given us, for the first time, a direct proof of the valdity of
Raper's scheme and the presence of a unit of hydrated indolquinone in the course
of melanogenesis.
In the animal kingdom there are black materials which originate from different
precursors of the tyrosine and therefore with a different melanogenetic process
like the porphirin-black,pterin- black, ommochrome-black, etc. Raper's scheme
seems to have a wide application in that the necessary tyrosine, tyrosinase and
O2 are highly diffused in the tissue.
From experimental and theoreical data collected to date it is possible to
propose a summary scheme for melanogenesis which adheres to experimental data
like that illustrated Tabella 2: Melanogenesis, Pheomelanogenesis,
Quinones hydrate.
Melanogenesis can be modified by cysteine. Lighter coloured materials
(red-brown, red, yellow) called pheomelanins form and are clearly visible in the
hair and skin of mammals and in the feathers of birds. These materials conserve
many of the properties of the melanins. In the drugged state they are amorphous
semiconductors characterised by a larger gap than that of the black materials
but less than that of yellow materials. The various tonalities and nuances of
colour, rather than a mixture of colours, may be induced also by electronic
transition values of band materials (see DISEGNO 3).
The colour of animals can be due to a physical effect (called schemochrome or
structural colour) or to a pigment. The blue colour is almost always a physical
colour known as the Tyndall blue or Tyndall effect. This colour does not alter
with the angle of vision. It is produced following the diffusion of the
radiation of wavelength less than white light during its passage through a
medium in which there is discontinuity: for example in the passage through a
colloid solution or a suspension of particles with a diameter less than a
twentieth of the wavelength of the incident luminous radiation. Beatiful
laboratory experiments ( Tyndall scattering or Tyndall blue,interference
colours, diffraction colours, colours modifiers,whitness,greyness ) are reported
in the Fox book
‘’The nature of Animal Colours ‘’ pag.183.
In 1869 John Tyndall was the first person to study the blue colour of the sky
and the phenomenon is often called the Tyndall effect to avoid confusion with
the diffusion that you have with the reflection of white light.
When the particles have a diameter less than the wavelength of red or yellow
light (a diameter of 0.6 mmicron) they can give rise to the phenomena of
reflection and diffusion of light in a greater measure for the shorter
wavelengths and lesser for the longer wavelengths.
The intensity of the radiation diffused by the very small particles, relative to
different spectral regions, is inversely proportional the the fourth power of
the wavelength of the incident light (Raleigh scattering). This means that the
radiations of the violet and blue of the spectrum are much more diffused than
the red ones. This is the reason that the smoke of a cigarette takes on a
sky-blueish colour against a dark background which impedes the reflection of
other colours which could mask the blue.
In 1866 Helmholz claimed that blue eyes are this colour because the colour is
derived from particles dispersed in a torbid medium on a dark background. Since
minute proteic particles, with refraction indexes higher than the surrounding
stroma, are found in the iris one retains that such particles spread the
incident white light giving rise to the blue colour. A gradual increase in the
dimension of the particles determines a decrease in the blue colour with age.
Besides, the dark brown pigment, precisely the melanin, which is found at the
base of the iris impedes sight of the red colour of the blood which flows in the
capilliaries. This phenomenon, however, occurs in the eyes of albinos who, as is
known, do not have the layer of melanin at the base of the iris. The granules of
melanin in the stroma at the base of the iris also cause the brown or yellow
colour of animal eyes.
In DISEGNO 2 there are three cases which we can denominate a, b and c from top
to bottom.
In case a the light is diffused by a particle of diameter less than 0.6 m(with
the production of a blue light which is visible with the screen or background of
melanin present in small grains which impede reflection of radiation of
different wavelengths. In this way the feather of a bird can appear blue.
In case b there is a yellow filter which is often constituted by a carotinoid.
One thereby has a green colour which we see in birds, feathers or in the skin of
tree-frogs.
In case c the black screen is modified, in that the granules of melanin are
concentrated in a point in the melanophores and the absorbtion of radiation is
diminished and the phenomenon of diffusion does not show the blue colour and
therefore the yellow colour of the pigment appears. The movement of the granules
can produce changes of colouration and thus in the appearance of animals for
example, cephalopods, reptiles and amphibians.
Structural colourations due to the Tyndall effect are amply represented in the
animal kingdom. A good part of the blues shown by animals have such an origin.
This is the case of the face and the posterior of mandrils, which highlight
regions which have a brilliant blue colouration, the scrotum of the cercopithecus
characterised by a light blue colour, the blue skin of the turkey and the neck
of the same colour of the pharoah hen, the blue of the African reptile Agama
cyanogaster and of numerous lizards. In all the listed cases the diffusion
is due to minute proteic particles which, when adherent to a underlying layer of
melanin, disperse blue more intensely.
Analogously the blue colour of the feathers of the kingfisher, of some varieties
of small bird, of parrots and many other birds is caused by the Tyndall effect.
To confirm the physical nature of the colour it is enough to observe a feather
under transmission of white light. When the colour is due to a physical effect,
the blue disappears and only a brown colour is seen. When, instead, the red or
yellow of a feather is due to a particular pigment, the colouration remains in
observation in transmitted light. The particles which determine the diffusion of
light in the feathers of birds are, generally, minute air-filled cavities inside
a layer of keratin called box cells.
One must pay attention not to confuse the colouration due to the Tyndall effect
with colouration due to interference phenomenon. This is the case, for example,
of a bird belonging to the Coracidii, precisely Coracias indica which has
blue coloured feathers which change colour with the angle of vision and become
green when immersed in water.
In fish, many teleosts present typical markings of a brilliant blue colour. Even
if in many cases the nature of these colours is not noted, that is, it is not
known if they are structural or due to pigments, in the Trachinids, for example
in Trachinus draco, and in the Gobiids, for example in Gobius
paganellus, the blue colour which distinguishes them seems to be
attributable to the Tyndall effect. In these fish light is diffised by minute
crystals or aggregates of guanin present in the guanophores superimposed on the
melanin of the underlying melanophores.
Again among the fish, precisely in the Dipnoi, the grey-blue appearance that the
Protopterus aethiopicus takes on approximately half-way through its
maturation process also seems to be due to the diffusion of light.
In the invertebrates the colours attributed to the Tyndall effect are quite
rare. They have been identified, though, in come dragon-flies belonging to the
order of the Idonati, precisely in the Escnidi and in the Agrionidi. In these
dragon-flies the characteristic metallic blue colour is due to diffusion of the
light in the epidermal cells which contain minute colourless granules deposited
on a layer of hommatine pigment of a dark brown colour with violet touches. In
particular
multidecker
graphite-like sandwiches (interspace 3.4 A°) or closed like the giant
fullerenes (interspace 4.5 A°). It would seem that in the eumelanins (see the
part on chemistry) the graphite type prevails and in the allomelanins the
fullerene type dominates. The cages of these materials can be broken up by
LASER, as for example that of the graphite which led to the discovery of the
fullerenes.The cages are sensitive to sonication.
The materials with band structure can be black, red or yellow according to the
gap (expressed in eV) of the prohibited band as represented in DISEGNO 3. All
the black materials are conductors, like the well known thiophene- black,
pyrrole- black, indole- black, acetylene- black, aniline- black etc.
The conductivity can be of the metallic type, semiconductor or superconductor
type (17) (23). They can give charge-transfer complexes and present the
phenomena of threshold switching (17g), (26), (27). They are sensitive to
radiation and transmit sound waves in a special way. The natural amorphous
materials (melanins) absorb ultrasound in the interval of 1 MHz both in vitro
and in vivo (27). The ordered polymers or non amorphous polymers or crystallines
in which there is the particular pseudo-conjugate system (28), (18), (19), (29),
show particular electrical properties like low energy optical transmission,
small gap of the band structure, low ionization potential voltage, and high
electronic affinity (23). Charge transfer agents and doping can convert an
isolating material into a material which is near some metals for its
conductivity property. These materials can conduct electrical current both in
suspension and in solution. The conductivity of the material is highly
influenced by the doping, by the solid state, by the oxydation state, and by the
hydration state of the polyquinone form. In the course of synthesis the pH, the
temperature and the concentration can influence the form and the size of the
particles and therefore the electrical properties of the material. The nature of
amorphous semiconductors poses new questions about the function of the melanins
especially in the eye, in the nervous system and finally in the skin as a
possible element of auxilliary communication with the CNS.
The electrical properties of the black particles, the phenomenon of threshold
switching, the photoacustic properties, the presence of stable unpaired
electrons (EPR), the modifiablity of the surface properties by physical and
chemical agents makes these molecules of great interest for biology in general
and for the physiology of vision, the substantia nigra, the communication
between tissues and the CNS (30) in particular. Under heating the black
particles lose water and carbon dioxide. Unfortuanately the incorrect method of
isolation and purification and the consequent degraded, heterogenous and
artificial material have not allowed, to date, obtaining satifactory results in
the study of the conductivity and of the other special physical properties of
the melanins.The electrical properties depend on the electronic transition
between bands and on the value of the gap of the prohibited band.
The colour of these materials depends on the transition between bands and on the
value of the gap of the prohibited band.
The
sequence of colours of the prohibited band, in increasing order by amplitude is:
black-- red-- orange -- yellow -- colourless
The
black materials are therefore characterised by a small gap.
The type of molecular and atomic assembly of the particles can influence the
colours.
In nature colours are attributed to different mechanisms of formation. Now, the
colours of the semiconductors are also considered.
The
situation for the attribution of colours can be summarised as
1. Vibration of the molecules (pure water).
2. Colour of the crystalline field (emerald, composed of transition metals).
3. Refraction, diffraction, interference (colour of the insects cuticle).
4. Diffusion of light giving Tyndall blue(sky blue, animal colours, eyes).
5. Transition of molecular orbitals (colourants, organic pigments)
6. Transition of materials with band structure (semiconductors, black / red /
yellow sulphides, melanins, pheomelanins, allomelanins, porphyrin iron salts,
salts of the complexes EDOEDTTTF e DOVDTTTF, e DOMDTTTF, MDTTTF).
The
coloured materials with band structures are much more widespread in nature than
is commonly believed (1a), (1g).Metallic or iridescent colours are observed in
some iron salts of the porphyrins (24) and in the complex salts of EDOVDTTTF
(ethylene dioxyvinylenedithiotetrathiafulvalene) (25).These composites are also
rare examples of black crystalline organic compounds.
The melanins have been the subject of many theoretical studies. (17), (31),
(32). The physical properties of these materials, for example the electrical
conductivity and the colour are in agreement both with the theoretical
predictions (Huckel calculations, ZINDO-CI calculations etc.), made to date, and
with the experimental data.
The melanins often have the physical-chemical characteristics required for the
realisation of superconductivity. They can be classified as amorphous
superconductors with threshold switching effect.
The black materials of TABLE 1 show stable unpaired electrons identifiable from
the EPR (electron paramagnetic resonance) spectra, a characteristic line of free
materials or of HLDS (highly localized defect states) and in general they have
all the requirements for a material to be electroactive. The characterstic
diffraction spectra at X-ray obtained from the DHI-melanin have been verified by
scanning tunneling microscopy (STM) in a Digital Instruments Nanoscope II at
room temperature (10). A computer elaborated construction of the formulae in
TAVOLA 2 e 3 has been made using the data obtained with X-rays and the data
obtained with the scanning microscopy.
Many blacks and the melanins behave as amorphous semiconductors TS (threshold
switching, communication at the threshold, relay effect) at potential gradients
lower than the inorganic films or other biological materials. This type of
electrical apparatus can involve skin, retina, brain and ear melanin (26).
The black materials can mutate their surface properties under the action of an
electrical field and the level of hydratation of the quinonic forms, can suffer
photolysis under the action of radiation.
The black materials explode and break up under the action of the LASER, in
pyrolysis, under atomic bombardment (FAB), and in cosmic collisions;the
sensitivity to radiation interests cosmochemistry (16).
All these properties are little influenced by the nature or the structure of the
melanogen used for the preparation of the black material.
In the course of studying the oligomer DHI(16) one obtains structures in which
porphyrine-like structures and heptagonal and octagonal rings are also
recognisable (10). The presence of a similar porphyrine system suggests that it
could also be possible to realise superconductivity through metallic complexes
or the superposition of porphyrin macrocycles. Besides is should be remembered
that in many porphirynic complexes of iron the colours seen (24) electric black,
blue-black, gold-black, copper-black are typical of the inorganic
semiconductors.
The
black materials are widespread in nature, both in the organic and the inorganic
world; and it is realistic that a great quantity of black material is also found
in interstellar space.
Chemical studies on the black materials of cellular origin (melanins,
eumelanins, pheomelanins, allomelanins) conducted in the last few years are
somewhat confused and little reproducible because they have worked on
heterogenous raw materials and on artificial materials. The most simple melanin
can be considered the acetylene-black from which it is possible to derive all
the others as illustrated in Tables 3 and 4. Substitution does not qualitatively
influence the physical properties like conductivity, colour, EPR, which remain
unaltered. The system indicated in red in the TAVOLA 3 can be taken as
indicative for a solid material with band structure.
The melanins are almost always cellular products. The melanins in mammals are
called eumelanins, the lighter ones pheomelanins, those of the vegetable world
allomelanins (2). The chemical knowledge of the natural melanins is still
limited to those of the indole polyphenols isolated in the course of the
melanogenesis of tyrosine.
The cellular natural black materials (melanins) are generally formed from
phenols, aminophenols and o.diphenols, the synthetic materials from different
substrates as reported in TABELLA 1.
Among these the DHI black recently identified with the melanin which is obtained
from the ink sack of the sepia Sepia officinalis, the dopa-black,
the dopamin-black, the adrenalin- black, the catechol-black and the
4-amminocatechol-black. For the melanins theoretical calculations and
experimental data indicate their nature of being amorphous semiconductors with
band stucture (18), (19), (22), (27), (31), (32), and particles with a fullerene
cage (4.5 A°) or graphite cage (3.4 A°). The cages of these materials can be
reversibly altered by ultrasound (15 min. at 80 W) [ (33) pag.103) ]: the sepia
melanin is soluble in alkaline pH and re-forms the originating structure in acid
conditions. Presumably in alkaline conditions the hydrated indolquinone
form predominates. The chemical and physical study of the melanin has also been
extended to materials of different origins like human hair, melanoma in rats,
dog hair and horse hair, in oxen eyes, and pigeon, chicken feathers the liver of
Amphiuma and of Axolotl showing their indole nature (34),(35).
The natural and synthetic black pigments are complex solid state materials.
Black pigments may be identified by cetoplasmatic formulae (18), (19). These are
limited to indicating the atomic skeleton of the melanogen, the presence of a
cation (anion) centre and of an unpaired electron that is, to indicating a
radical-polaronic system. The black materials are amorphous and soluble and
purifyable with difficulty. On heating the melanins lose water and carbon
dioxide. The loss of H2O can derive from the hydrated form of the quinone
(reversible H2O) or from the synthesis of oxygen bridges (irreversible H2O). The
CO2 can come from preexisting carboxylic groups -COOH or artificial derivatives
of the fission of the indole benzenoid part.
These materials bond acids and bases by chemical reactions of salification or as
coordination complexes like porphyrin or because of interstice phenomena.
Interstitial compounds can also form with gases in the proposed structures in an
analogous way to what happens with the fullerenic or graphitic carbons.
These materials are sensitive to light, to pressure, to oxygen and to peroxide.
The oxygen reacts with the hydroxyls of the DHI with the formation of H2O2 and
quinone systems; the hydrogen peroxide reacts in turn, breaking the C---C link
at the level of the hydroxyl groups. The black material formed by
5,6-dihydroxyindole (DHI-melanin), like all the blacks obtained from o.diphenol
is easily oxidisable. It is often reported in the literature that the melanins
are polyindolquinones but the centesimal analysis of the melanins are in
disagreement with the theoretical values calculated for a polyindolequinone
structure:
Found
for DHI-melanin : C% 56.6 H% 3.1 N% 8.2
Calculated for C8H3NO2 : C% 66.2 H% 2.1 N%
9.6
Calculated for C8H5NO3 : C% 58.8 H% 3.0
N% 8.6
Calculated for C8H7NO4 : C% 52.4 H% 3.8
N% 7.7
These
discordant values cancel each other out if one considers one or more quinone
carbonyls in the hydrate form (in such a form the molecules would no longer be
toxic for the cell). The hydrate polyindolquinone systems, new structure in the
chemistry of natural products, oxidise very easily with the opening of the
benzenoid ring. It is probable that with an analogous mechanism the melanins are
metabolised in organisms (oxidative phagocytosis) (41).
The black materials (melanins) react with alogens, CH2N2, H2O2, diazonium salts.
They also bond to the ions, to dopants, to water and to gases. The absorbtion of
gases on the part of the graphitic and fullerene systems is well known. Hydrogen
peroxide or the KMnO4 attaches melanin in alkaline conditions solubilising it.
Hydrogen peroxide, well known in the mass-media for its use in the tinting of
hair, acts on the sepiamelanin, the melanin contained in the ink sack in a
particular way. One observes a rapid solubilisation which corresponds to the
break up of the graphite cage followed by a slow transformation into a
yellow-golden solution. In the initial phase of the process it is possible to
obtain a black or red-brown pigment 60%-70% yield (according to the time of
action of the hydrogen peroxide) soluble in alkalis and precipitable in acids
called sepiomelanic acids (40). Such acids form because of the partial opening
of the indole benzenoid part. In the first phase proteic materials also appear
in quantities in the order of 4-5% for sepiomelanin (for melanins from other
sources 10%). It is uncertain if the proteic material is chemically linked to
the pigment or not. In contrast to the general opinion that the melanins are
melanoproteins is the fact that the melanosomes (hair, melanomas, eyes,) treated
with concentrated HCl for 24h 110° seem to remain unaltered conserving
form,size and fine structure (41).
The lack of hydrolisable links in the granules leaves one to suppose that among
the melanins and proteins there is a sort of sclerotisation as happens in the
cuticle of insects with the formation of non-hydrolisable links, or that the
protein is only mixed with the melanin (as is known a protein with enzymatic
activity is found in the sepia ink sack). The degredation of the melanosomes
which occurs in vivo due to the lisozomes is not due to an hydrolythic process
but to another oxidative rupture of the links at the work of a peroxydase type
enzyme.
The yellow-golden solution which is obtained by forced oxidation of the
heterogenous material extracted from the sepia ink sack contains a lot of oxalic
acid and a mixture of pyrrolic acids among which predominantly the acid
2,4,5-pyrroletricarboxylic and pyrrole complexes among which an acid isolated as
Ba salt (40) (42) of the formula C20H11N3O15Ba3
(this interesting compound has not been further examined). It is not possible to
assign a particular structural and analytic meaning to these demolition products
since they are obtained from samples of heterogenous and artificial materials in
the course of extraction and purification. Partial hydrolyses are operated by
the acids and the strong bases. Hydrochloric acid links to the melanin with a
process of addition and substitution still unknown. Concentrated HCl can still
operate the formation of oxygen bridges, esterification, polycondensation
(examples are the formation of carbazole structures from indole, and furane
structures from oxyindoles).
The diazomethane and the dimethylsulphate react with the melanin. The structure
of these derivatives so obtained are in agreement with the quinone hydrate form
of melanin. From literature data it appears that tyrosine-melanin also is
largely in hydrate form (49). The centesimal analysis (36) of the DHI-melanin
(eumelanin) gives values in accordance with those theoretically calculable for
the polymers of DHI only if this is thought of as a hydrate. The reversible
reaction of hydratation can be imputed to a reaction of addition of water to a
quinonic system (Table 3 and TABLE 2). One could think to an anlogous mechanism
for the hemicellulose and the collagen (30), (37), (38), (39).
This mechanism can also involve the electric phenomenon of threshold switching
or Proctor McGinness effect which is often observed in the hydrated black materials and may also be in
relation to the capacity of the melanin to mutate its surface properties under
the action of external forces like for example electrical, electromagnetic and
photoacoustic fields. The DHI-melanin examined at X-ray gives diffraction
spectra which agree with the polycondensed and polymerised systems assembled in
graphitic sandwiches and with the presence of porphiryn-like centres and
heptagonal and octagonal rings (10)..
It has been suggested that the assemblage of the atoms of C, H2O, of proteins,
virus, cells follows the architectural principle called tensegrity (39). The
melanin is a universal geodetic material sensitive to light to oxygen and to an
electromagnetic field. Such material is able to initiate, catalyse and influence
numerous chemical reactions including those at the origins of the first
molecules of assemblage.
The property of the black substances of breaking up under the action of
radiation, electron and atomic bombardment is interesting. The black material of
the Universe can break up, according to a process reproducible in the
laboratory, with the formation of simple organic molecules which are considered
as the building bricks of life (12),(14),(15),(16).
The quinonic hydrate form can, besides, mean the fixing of water to be easily
transported in a different chemical and solid form (earth, meteorites, comets).
One can, for this reason, suppose that biological evolution could be initiated
on a multifunctional film of melanin rather than from an anonymous biological
soup. The electrical and phonic conductivity, the capacity to bind solid, liquid
and gas products, the capacity to give quinonic hydrated forms, are properties
which make the black materials very interesting..
Melanins interest biology because they could intervene in the control of the
form and of the functioning of cellular adherence. As amorphous semiconductors
and superconductors they could constitute a system of auxilliary communication
in the tissues and for the CNS.
The melanins in the hydrate polyquinone form have a framework with a bed of
hydroxyls where water can be given up or taken up under the action of external
physical and chemical stimuli. The framework of the system is easily removable
by peroxydation.. On this framework the cells multiply until they form a
functioning organ.. Because of their chemico-physico polyfunctionality, melanins
are theoretically better adapted than PLA (polylactic acids) and PGA
(polyglycolic acids) and PLGA (PGA+PLA) or even than the so-called collagen
sponges+PLGA used for cellular assembly (30), (39).
For
many melanins the hydrate quinone structure is evident from:
The C, H, N centesimal analysis.
The IR spectrum.
The MALDI and MALDI-TOF spectra.
The MALDI-TOF spectra of the intermediate melanogenesis (13).
The REACTION with diazomethane and dimethylsulphate (2c).
A
quinone-H2O equilibrium may not only be an important transformation
for the cellular organisation and reorganisation but may also represent the
modification of physical properties (conductivity) and chemical properties
(solubility and reactivity) of the melanin. A consequence among others is that
the conducibility of the melanin must be measured in the natural hydrated form.
Every distortion which opens a gap in the density of state of the whole Fermi
surface can induce a transformation of the metal-insulator phase which is seen
with the change of colour. The relationships between the chemical variations of
the surface of these materials with the conducibility and with the various
phenomenon connected to it remain to be established. Associating the melanin to
fibres of the liquid crystal collagen (30) one can have an exaltation of the
assembling properties of the melanin. The electrical conductivity can furnish a
capillary intercommunication system in the tissue, the so called conscience of
the tissue.
It is reasonable to think that in the physiological conditions the conscience of
the brain and that of the tissue inform each other reciprocally through the
melanin: the tissue starts from the brain and the brain starts from the tissue.
The samples of melanin to study have to be made up of particles homogenous for
form and density (to be found from one time to the next) and prepared at room
temperature. The sonication can be used to dissolve melanins. One must avoid
contact with light, with atmospheric oxygen and dissolved oxygen, the use of
strong acids and bases. The use of chemical oxidants should be rationalised.
All the black materials, in particular the melanins, have been obtained or
treated without taking into account their real nature. It is necessary that in
the future results obtained are critically and rationally evaluated.
The case of sepiomelanin is relevant.The most studied natural black substance,
for the ease of access to it at the biological source, is the melanin which can
be extracted from the ink sack of the sepia and is called sepiomelanin. The
extraction of sepiomelanin presents great difficulties not only because of its
insolubility.
The ink of the sepia is in fact constituted by a mixture of melanosomes,
premelanosomes, (in which oxidative enzymatic activities are in act with the
rearrangement, transferring and elimination or creation of atomic and radical
groups) and granules of stabilised pigments. One thinks of diffusion and
therefore of the presence of enzymes like laccase, tyrosinase, peroxydase,
tautomerase (48). And for this reason it is possible that, in the mutated
environmental conditions verified in the course of the extraction, the cellular
process is modified with the formation of a melanin which is, effectively,
artificial. The principle modification would seem to operate at the level of the
dopachrome (2e) (8) with the formation of DHICA instead of DHI. That would lead
to the description of a melanin formed in the prevalence of DHICA units instead
of DHI contrary to that predicted in the scheme of the melanogenesis and by the
activity of the tautomerase dopachrome (8a). Another collateral process can be
that of the opening of the benzenoid rings by hydrogen peroxide (a normal
product of melanogenesis).
On the basis of the chemical and physical data available to date it is possible
to conclude and summarise:
The melanins are polyquinones in a hydrate form.They are characterised by a
radical-polaron system with stable unpaired electrons. Such a system is present
in DHI-melanin and in all the organic black materials examined. The oligomers
(12-16 monomers) are settled in graphitic sandwiches (interspacing 3.4 A°) or
in fullerene cages (interspacing 4.4 A°). The melanins are natural amorphous
semiconductors with a model which corresponds to the band model of that of
semiconductors and superconductors.
On a biological level the black particles theoretically possess multi-functional
properties yet to be discovered, like the capacity of molecular
synthesis, of molecule and cell assembly, the function of communicating between
tissue and the central nervous system, the storage of water, metals and gas. The
particles of melanin explode under the action of the LASER and atomic
bombardment continually transforming in cycles of synthesis and of break up
(both on earth and in interstellar space).(12), (14), (15).
-------------------------------------
O.Diphenol
formation from phenol without enzyme.
From
W.Brackman, E.Havinga, Rec.Trav.Chim.des Pays-Bas 74,1107, (1955).
It was found that the primary reaction consists in the
introduction of a hydroxyl group into phenol.This reaction occurs within a
complex of copper with morpholine, phenol and hydrogen peroxide. The catechol
formed is oxidized further either by oxygen or by a cupric-morpholine complex to
give ortho-benzoquinone. This takes up morholine to give the morpholinocatecol,
which is subject to a rapid autoxidation to a morpholino-ortho-benzoquinone. The
addition of a second morpholino molecule followed by another autoxidation gives
rise the main reaction product: dimorpholino-ortho-benzoquinone.The hydrogen
peroxide necessary for the primary attack upon the phenol originatfrom the
autoxidation of the catechol formed. The primary reaction occuring in the
hypothetical copper-morpholino-phenol-hydrogen peroxide complex explains the
specific ortho-oxidation as well as the
exclusive activity of copper in these catalytic oxidations.
-----------------------------------------------------
Sample
preparation of melanin from biological sources based on laboratory experience
with Sepiomelanin
The
preparation of samples of melanin regards, for the moment, only melanin from the
sepia. In a similar way would be possible to obtain samples of melanin from
different biological sources.
The sepia ink is a complex mixture of organelles, premelanosomes, melanosomes,
granules, proteic material (enzymes), glucosamine, and phospholipids in
suspension or solution liquid. At the moment of extraction the mixture is still
active and contains some hydrogen peroxide. An artificial melanin, that is a
chemical product different to the physiological one with a possible formation of
a system built on units of DHICA rather than DHI, may be formed.
The composition of the mixture is very variable according to whether one is
dealing with the ink of a live animal or a dead one, and on the time spent
between one emission and another of the black of the animal. For this reason the
goal of obtaining a reproducible sample is difficult and laborious to reach. The
main problem is to use samples almost formed of granules and material not
contaminated of hydrogen peroxide.
Samples obtained from naturally fresh ejected ink are recommended or to proceed
in the following manner :
Sepia was killed with urethane.The sepia ink pouch is opened and the liquid
gently squeezed out. To the black suspension catalase ( amount to be defined )
and water (20% distilled, deionizated and deoxygenated water) is added and
centrifuged at 2000-3000 cycles. The black solid is washed with H2O
x3, CH3COCH3 x3, H2O x3, and dried on KOH
pellets. All the operations are conducted at room temperature and away the
contact of light and as much as possible away from atmospheric oxygen.
The black solid thus obtained is rich in ashes (Na, K, Ca, Mg up to 20-25%
expressed in sulphates) and contains about one oxygen atom for every IQ unit (
addition of water to quinone group, storage of O2, water,presence of
carboxylic groups )
This sample (A) called sepiomelanin is a salt, the Mg and Ca salt, and can be
used in the same way either in the form of a free acid treating it with HCl 2N
x3, H2O x3, in centrifuge, obtaining (B), the sepiomelanic acid.
The
sepiomelaininc acid (B) can also be obtained by the following method:
The solid (A) is suspended in 80 cc of H2O and taken to pH 10 by adding NaOH N,
passed through ultrasound (15 min 80W) and eventually filtered or centrifuged.
The filtrate is taken to pH 1 with concentrated HCl and the solid centrifuged
and washed with HCl N x3, H20 x3, acetone x3, H20 x3,
dried on KOH drops at room temperature and away from light.
Both
sepiomelanin and sepiomelaninic acid can be further purified using various
methods (43).
Samples of different composition can be obtained varying the speed of the
centrifuge. Using MALDI mass spectrometry and MALDI-TOF on these samples it is
possile to carry out an in depth examination of the process of melanogenesis
which happens in the ink sack of the sepia Sepia officinalis (5).
Summing up: successful work needs a homogeneous preparation of granules,
working when possible in the absence of hydrogen peroxide ( peroxide is present
in the cell or formed by action of atmospheric oxygen on o.diphenols ) ,light,
oxygen, at a physiological pH. The samples for centesimal analysis must be dried
at room temperature.
The samples undergoing analysis must give values of C, H and N taken from the
ashes, in agreement with the theoretical values calculable for a
polyindolequinone hydrate (various quinonic structure). Typical broad IR, 13C
NMR, MALDI spectra are to be compared
with DOPA-melanin and DHICA-melanin.
Extraction of melanin from biological sources
The
dried fresh material was treated with 5% ammonia solution and few mg of sodium
bisulphite.Sometimes sonication ( see sepiomelanin) is necessary.The cooled
yellow-brown solution was acidified with conc. hydrochloric acid to Congo red ,
centrifuged and dried at room temperature.
IR,
MALDI, 13C NMR spectra, analysis for C, H, N, S, Fe, Cu, Ni, Cd, C
are compared with those of DOPA-melanin and DHICA-melanin.The storage of gases
and the bindig of foreign substances are determined .
Oxidative degradation of natural pigments :
identification of 2,3,5-pyrroletricarboxylic acid
(micro-equipment
necessary)
Samples
of natural pigments (25mg) were dissolved or suspended in 2n potassium carbonate
(2ml) and oxidized at room temperature by the gradual addition of saturated
potassium permanganate solution.When the colour of permanganate persisted for
about 10 minutes excess of the oxidant was destroyed by
addition of a little sodium sulphite.The solution was briefly boiled and
freed from manganese dioxide by filtration or centrifugation.The manganese
dioxide was washed with hot distilled water (3ml)the washing being added to the
main filtrate . The combined filtrate and washing,acidified to congo red and if
necessary filtered was adjusted to ph 4-4,5 by addition of 2N NaOH .
50% Calcium chloride solution (1-2 drops ) was added and a precipitate
forming during 1 hour was removed by filtration or centrifugation. After
ensuring that a portion of the solution afforded a precipitate with ammonium
oxalate solution it was made strongly acidic to congo red by addition of
conc.hydrochloric acid, pyrrolic acids were now extracted with peroxide-free
ether ((4x2.5ml). The ethereal solution was washed with distilled water (0.5ml)
dried over magnesium sulphate and evaporated to 2-4ml in vacuo.Finally
evaporation to dryness was effected in a small text tube at 60-70°.
To the
residue distilled water (0.1-0.2 ml.) was added and the solution was filtered
through cotton wool (see Figure).The straw yellow filtrate was stored for 12
hours in a small test tube (5 mm. x 3 cm.) and was then filtered for a
second time. .
The
perfectly clear solution was used for chromatography on Whatman n°1 paper in
comparison with, and in mixture
with authentic pyrrolic acids . Such solutions
provided sufficient material for several chromatograms. To ensure
identification of acids the
chromatograms were developed in a number of
different solvents 12 hours being necessary
for good separation of acids. After drying , the
papers were sprayed
with a freshly
prepared solution of diazotized sulphanilic acid (solutions of 0, 2 g. of
sodium nitrite in 35 ml. water and
0,5 g . of sulphanilic acid in 35 ml. of water
containing 1, 5 of N caustic soda mixed and made just acid to Congo red by
the addition of conc. hydrochloric acid) and subsequently
with N caustic soda.
This
treatment caused the appearance of intensely
coloured spots. The colours slowly
faded but could be restored by further spraying
with caustic soda. Thereinafter
the diazonium salt together
with the caustic soda reactant is referred to as
DZA.
Synthesis of pyrrole-2,3,5-tricarboxylic acid
2,5-Diformyl-3-
chloropyrrole-4- carboxylic acid (0,9 g.) (Fischer, Sturm, Friedrich, Ann.
461,260, 1928 ) was dissolved in the smallest quantity of 2N Na2CO3
and oxidised by the gradual addition of a 5% potassium
permanganate solution.The temperature was kept at about 25-30° by occasional
cooling in water.
When
the colour of permanganate persisted for 30 min. a small amount of sodium
sulphite was added. The resulting
mixture was boiled, the MnO2 filtered
off and washed several times with hot water. After cooling
the combined filtrate and washings were strongly
acidified with conc. HCl. A
crystalline precipitate of the sodium hydrogen salt of the acid
was sometimes formed. The solution was extracted
repeatedly with ether. The extracts after drying over sodium sulphate,
were evaporated to dryness leaving
a yellow solid deposit of 3- chioropyrrole-2,4,5-tricarboxylic acid
Crystallization of the crude
product from dioxane (charcoal) yielded 150 mg. of colourless needles m.p. 300°
(dec.).
Raney
Ni ( 500 mg ) was added to a solution of 3-chloropyrrole-2,4,5-
tricarboxylic
acid ( 150 mg ) and NaOH (120mg) in water (12ml).Reduction was carried out in
hydrogen for two hours (80°/50 Atm.). After
cooling the catalyst was filtered off, the solution acidified to Congo red and
the product extracted with ether ( 300 ml in 6 portions ) .Distillation of ether
gave 50mg of pyrrole-2,4,5-tricarboxylic acid after recrystallization from CH3COOH
( melting point 295° dec. ).Sprayed with DZA on paper it gave a deep red
fleck.
Others laboratory experiments
For experiment on green,
blue, iridescence, interference, refractive index of colours see Fox and Vevers
‘’ The Nature of Animal Colours ‘’
Sidgwick and Jackson, London 1960, pag.183-209
LEGENDA:
(Drawing =Disegno ; Photo = Foto ;
Table = Tavola).
TABLE
1
list of black materials.
TABLE
2
Melanogenesis, Pheomelanogenesis, Quinones hydrate.
PHOTO
1
The photograph shows two guinea pigs (Cava cobaya) one black, one red.
The black fur comes from DOPA while the red comes from cysteinildopa.
PHOTO
2
The multi-coloured Bengal Pitt. Melanin is an indispensable material for the
vision of the blue and the green and their shades.
PHOTO
3
Two tree-frogs (Hyla arborea) one green and the other light blue. If the
green frog lacks its yellow pigment because of a genetic mutation it is blue.
DRAWING
1
Melanosome of the Harding-Passey tumour at different stages of development.
DRAWING
2
In Nature there are not blue and green pigments. The picture illustrates how the
colours blue, green and yellow can be generated in animals.
DRAWING
3
The semiconductor band structure attributed to the melanins. The melanins can be
considered to have band structures with a small gap.
EG = Optical band or gap (colour band), Fermi prohibited band. The passage of
the electrons from the valence band to the conduction band is facilitated by the
temperature and by dopants. The amplitude of the gap determines the colour, the
conducibility or insulating state. In amorphous materials the same model as for
the semiconductors is accepted even though the theory is still qualitative at
the electron level.
BW =
Width of the valence band
EA = Electrical affinity. A high value indicates an easily reducible material.
It is measured from the base of the conduction band to the vacuum.
IP = Ionisation potential which measures the energy to remove an electron of the
material in vacuum. A small value indicates an easily oxydisable polymer.
TABLE
1
Products of the tyrosine melanogenesis. Cyclodopa and not DHICA is probably the
precursor of eumelanins Among the intermediates only DHI and DHICA have been
isolated in the crystal state and adequately characterised.
TABLE 2
The acetylene black, the simplest of the radical-polarons present in many
materials.
The illustration shows a unit of 16 DHI monomers assembled in a graphite
sandwich seen in MALDI and X-ray spectra (5) (10). The computer elaborated
formula has interesting porphyrine sites and octagonal rings which explain the
notably capacity of DHI-melanin (sepiomelanin) or black materials in general to
bind solids and gases.
TABLE 3
The characteristic structure of black band materials is shown in red.
The hydrated form of indolquinone is non-toxic for cells and possesses many of
the surface properties of the PLGA-collagen. Due to its electrical properties it
represents an element of communication between the tissues and the CNS.
In humic acids the reversible hydrated form can be a reserve of water to be
redistributed in the ground. This solid water could be transported in similar
forms by comets and meteorites.
The C, H, N centesimal analysis (36)carried out on correctly treated samples of
DHI-melanin (sepiomelanin) gives values which, subtracting the ashes, are in
close approximation to those calculable for a hydrated polyindolquinone.
The analytical data are confirmed by the study of dopamine and DHI melanogenesis
by mass spectrometry (13). MALDI spectra show peaks of oligomers
fragmentation.
-------------------------------------------------------
Dopachrome
does exist ?
DOPA ® Dopachrome---- DHI ® IQ ®
PIQ hydrate form----- (black guinea-pig).
DOPA + cisteina ® Cysteinyldopa-----
(CAVIA ROSSA red guinea-pig ).
Dopachrome(
name given to a red solution probably a mixture of oligomers ) may
be not decarboxylated and DHICA is formed.Oxidation of DHICA gives a brown
carboxylated material .
DHI = 5,6-dihydroxyindole
DHICA = 5,6-dihydroxyindole-2-carboxylic acid
P = Poly
Q = Quinone
I = Indole
DISEGNO
1. Melanosomi del tumore di Harding-Passey a diverso stadio di sviluppo
BLUE
GREEN
YELLOW
DISEGNO 2. In Natura non vi sono
pigmenti blu o verdi. Il DISEGNO illustra come si possono generare i colori blu,
verde, gialli negli animali.
EG = Banda ottica o gap (Banda dei colori), banda proibita di Fermi. Il
passaggio degli elettroni dalla banda di valenza a quella di conduzione è
facilitata dalla temperatura e da tracce di impurezze (droghe) L'ampiezza del
gap determina il colore,lo stato conduttore e quello isolante. Nei materiali
amorfi è accettato lo stesso modello del semiconduttore anche se la teoria dei
livelli elettronici è ancora allo stadio qualitativo.
BW= larghezza della banda occupata di
valenza
EA = Affinità elettronica. Un valore
elevato indica un materiale facilmente riducibile. Si misura dal basso della
banda di conduzione fino al vuoto.
IP = Potenziale di ionizzazione che
misura l’energia per rimuovere un elettrone dal materiale nel vuoto. Un
piccolo valore del potenziale indica un polimero facilmente ossidabile.
Drawning 3. La struttura a bande del
semiconduttore attribuibile alle melanine.Le melanine si possono considerare
materiali a bande con piccolo gap.
PHOTO 1. La foto rappresenta una cavia (Cava cobaya) nera ed una rossa. Il
pelame nero si origina dalla DOPA mentre quello fulvo dalla cisteinildopa.
-
Pigmento giallo + Tyndall blu
- Melanina
- Tyndall blu
- Feomelanina
PHOTO
2. Il variopinto Bengala Pitt La
melanina è un costituente indispensabile per la visione del blu e del verde e
delle loro sfumature.
PHOTO 3. Rappresenta due raganelle
(Hyla arborea) una verde e l'altra azzurrina. La rana verde perdendo il suo
pigmento giallo è diventata azzurra per una mutazione genetica.
TAVOLA 1. Prodotti della melanogenesi da tirosina. Fra gli intermedi solo il
DHI e il DHICA sono stati isolati allo stato cristallino ed adeguatamente
caratterizzati.
Il
nero di acetilene il più semplice dei radical-polaroni presenti in molti
materiali neri.
Si mostra una unità di 16 monomeri di DHI assemblata in sandwich grafitici
ricavabile dagli spettri MALDI e da quelli a Raggi-X (10). La formula dedotta al
computer mostra interessanti siti di tipo porfirinico ed anelli ottagonali che
rendono conto della notevole capacità complessante di solidi gas presentata
dalla DHI-melanina (sepiomelanina) e dei materiali neri in generale.
TABLE
3. In rosso la struttura caratteristica dei materiali a bande neri.
La forma idrata dello indolchinone è atossica per la cellula e possiede molte
delle proprietà di superficie del PLGA-collageno. Per le sue proprietà
elettriche rappresenta un elemento di comunicazione fra tessuto e SNC. Negli
acidi umici la forma idrata reversibile può rappresentare una riserva di acqua
da ridistribuire nel terreno. In forma simile questa acqua solida potrebbe
essere trasportata da comete e meteoriti.
L'analisi centesimale (36) C, H, N eseguita su campioni di DHI- melanina trattati
in modo corretto dà valori. che, sottratte le ceneri, sono in buona
approssimazione con quelli calcolabili per un poliindolchinone nella forma
idrata.
I dati analitici sono confermati dallo studio della melanogenesi della dopammina
e del DHI con l'ausilio della spettrometria di massa (13). Gli spettri MALDI ,
eseguiti nel corso del tempo della melanogenesi , mostrano picchi non solo
relativi ad oligomeri del DHI ma anche a quelli dei suoi derivati ossidrilati.
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_______________________________________
Taken
in part from R.A. Nicolaus, G. Parisi '' The Nature of Animal Blacks". Atti
della Accademia
Pontaniana
Vol.XLIX. and Link 9 of www.tightrope.it/nicolaus/index.htm
See
also Link 1.
for
correspondence and comment: rnicolaus@tightrope.it
- parisi@cds.unina.it
Accademia Pontaniana,Via Mezzocannone 8, I-80132, Napoli.
http://www.
pontaniana.unina.it
Naples Novembre 1999
Revised December 2003-12-16