COLUMBIA - A joint House-Senate ethics committee began initial discussion Tuesday on a sweeping ethics reform bill as lawmakers try to beat the clock to get a bill passed before the end of the legislative session this week.

The two versions setting up new rules for lawmakers' conduct vary widely, and both sides said they would talk to their colleagues to get a sense of what each body is willing to give.

The House version establishes a 12-member independent commission that would be able to investigate members of each branch for ethics infractions. That body would investigate ethics-related allegations in all three branches of government, but disciplinary decisions would be left up to the current bodies that decide those matters. The bill places restrictions on appointing campaign donors and family members to that committee.

The House bill reconstitutes the State Ethics Commission, which oversees executive branch officials, including the governor. As it stands, all nine members of the commission are appointed by the governor, but the House bill would spread appointees among other statewide offices.

Sen. Wes Hayes, R-Rock Hill, who chairs the joint ethics panel empowered to come to a compromise between the two bodies, said that the independent commission was likely a non-starter with much of the Senate. The Senate already rejected the concept when it passed its version of ethics reform earlier this year.

He said he was focused on a potential compromise including what he calls the "big three" issues: assuring lawmakers disclose their sources of income; requiring groups and Super PACs that spend money on campaigns in South Carolina to disclose their donors; and banning so-called Leadership PACs, lawmaker fundraising groups that raise funds and then spend dollars without the oversight and scrutiny of campaign funds.

"We'll have a bill and hopefully have the votes to pass it," Hayes said.

Rep. Bruce Bannister, R-Greenville, who is on the joint committee, said he would poll members of the Republican caucus Tuesday to see what was possible.

"If the independent investigation committee is a non-starter (with the Senate) do we want to go forward?" Bannister said. "I'd like to see us do something."

Critics have already knocked the bill for its failure to ban or limit the use of campaign funds for office uses, a source of much controversy recently. The House bill also allows some sources of income for lawmakers and family members to remain out of public view, something advocates say could provide future loopholes.


Police officers search parts of the San Diego river for homeless people who have taken up residence near the waterway. Officer John Horvath wakes a sleeping transient who had an outstanding arrest warrant on his record.

— From the sidewalk of Friars Road in Mission Valley, the San Diego River can be seen slowly snaking through tall grasses.

It’s a historic, 52-mile slice of nature, home to furry denizens and explored by hikers and wildlife enthusiasts.

But others make the river’s shores their home. Drug addicts. Thieves. The mentally ill.

A persistent homeless population has laid its claim on the environmentally sensitive zone for decades, polluting the area with trash and human waste. San Diego police officers and a homeless outreach team visit the area at least once a month, but the people who call the river home often don’t want help and create problems in surrounding communities, a lieutenant said.

“This is a huge issue for the community and they’re screaming about it,” said police Sgt. Jack Knish.

Officers raided one of the riverside encampments, then known as Edge City, as early as 1987, and have policed the territory ever since. Cleanup efforts are just as frequent. Trucks of trash are regularly hauled away. The water itself, visibly polluted, is harder to clean.

The river’s shanty towns can at times be sophisticated, complete with stoves and appliances that run on stolen electricity. Zones have even been given neighborhood-like monikers by homeless residents and police alike — Mt. Trashmore, The Pit, The Dungeon and Gilligan’s Island.

While it’s not a crime to be homeless, many who take up long-term residence are involved in criminal activity, police Capt. David Rohowitz said. One island resembles a bicycle chop shop. Gears, chains and other parts are organized in piles to be hocked later. Police field calls about the homeless victimizing pedestrians, breaking into cars and committing robberies. Arson is another persistent issue.

Even more often, police are sent to the riverbanks to address violence committed by one homeless person against another.

“Many are on probation or parole with psychological issues or drug or alcohol addictions,” Rohowitz said. “They typically aren’t balanced people down on their luck. These are people on the margins of society.”

Controlling criminal activity poses challenges. Transients are venturing deeper into the brush, making it more difficult for patrols to contact them. Issuing citations that stick is even harder.

When officers run into someone living in the area, they check for outstanding arrest warrants. Police tell the riverbed resident that no one is allowed to live there and offer services from the department’s Homeless Outreach Team.

If patrols come upon that person again, they dole out a citation for illegal lodging. If, after a citation, homeless people still stick around, they can be arrested. Multiple arrests could lead to a stay-away warrant, barring that person from the area and resulting in a longer jail stay.

Even after all these deterrents, too frequently, they head right back to the river, Knish said.

“The person who lives in the riverbed knows that nothing is going to happen to them, so they say, ‘You can arrest me all day long, I don’t care,’” he said.

Without more long-term consequences, the river population will likely stay steady, Knish said.

Sgt. Teresa Clark, who manages all of San Diego’s homeless outreach teams, said the river’s residents tend be a more hard-core group of transients.

“There are more veterans, more parolees,” she said. “They are rougher and are better at roughing it.”

The sergeant said it’s a difficult group to work with and one that rarely wants help, but that doesn’t stop the department’s homeless outreach teams from offering. Working with the county’s health and human services department and the psychiatric emergency response team, officers are able to help homeless fill out affordable housing forms, get identification and connect with other social services.


12 November 2012

Google TV, accessed via a set-top box or integrated into the latest HDTVs from Samsung, Sony and LG, is designed to combine the best of the internet and Android plus the Google Play app store, with the best of TV. Viewers can surf the web, find programming based on themes, genres and likes, and download and install apps to improve the viewing experience. However, due to international licensing and rights laws, two of Google Play's most attractive features, downloading and streaming video and music content, have previously been unavailable to users outside the US.

From November 13 that is set to change with the UK, France and Germany becoming the first outside America to be able to access everything. However, unlike in the US, they will not be able to download the content to other Android devices such as tablets or smartphones, though it is hoped this limitation will also soon be addressed.

Amazon faced similar issues when it launched the Kindle Fire HD. Despite being able to draw on a huge ecosystem of multimedia content, the company hadn't negotiated cross-border licensing with the rights holders, which is why the original Kindle Fire was released in the US only. The company has since agreed deals with all of the major record labels and film studios, which means it has been able to launch the second generation of its 7-inch tablet internationally.


Have we just seen the turning point in equity markets? A slew of bearish data from the US includes renewed predictions of falling property prices, and the UK should ensure credit is available to non-financial institutions too, if it wants to avoid the fate of mid 1990s Japan Read more

Oil trades may be denominated in a basket of currencies, rather than the US dollar. Brazil is having a good week, and there are growing calls for IMF reform to go further than that agreed at the G20 Pittsburgh meeting Read more

“Well run” emerging economies will be encouraged to hold less in foreign reserves with the IMF acting as a giant insurer, under a new proposal. Unemployment is set to break through 10 per cent in the US, and stay there for quite some time Read more

China’s push for materials and energy leads it to be South Africa’s biggest trading partner. In spite of this, questions over China’s continued recovery and further questions on whether we are seeing the end of the rally in equities Read more

As Russia invites foreign investment, China encourages domestic investment in commercial property. US housing prices are marginally improved but on thin volume, and unemployment data looks grim Read more

As money pours out of money market funds, we ask whether equity levels are sustainable and if the return of property-backed debt is a cause for celebration. Food security is a rising international concern and Bill Clinton asks if we should use the unemployed to increase energy efficiency. Read more

Jobs data looks healthy in Brazil and the square mile, but less rosy for manufacturing workers and journalists, although wage data may be good for those employed, whatever the sector. Commodities are ever more popular and gold could surge if China bans exports Read more


Be Sociable, Share!

    What is strabismus?

    Strabismus is a condition in which your eyes are not properly aligned with each other, resulting in double vision or the suppression of the image from the affected eye. For a variety of reasons, one or both of your eyes may turn in, out, up or down.

    What causes strabismus?

    Coordination of your eyes and their ability to work together as a team develops in your first six months to four years of life. Failure of your eye muscles to work together properly can lead to strabismus. Strabismus can be hereditary, but may also be acquired secondary to an eye injury or disease.

    Who is affected by strabismus?

    Children under age six are most affected by strabismus, but it usually first appears between birth and 21 months. It is estimated that five per cent of all children have some type or degree of strabismus. Although rare, strabismus can sometimes begin in adulthood. Sudden onset of strabismus may occur as a result of a stroke, tumor or other vascular disease.

    Will a child outgrow strabismus?

    A child will not outgrow strabismus without treatment. In fact, the condition may simply become worse without treatment leading to an amblyopic (lazy) eye.

    What are the effects of strabismus?

    Children with strabismus may initially have double vision. This occurs because both eyes are not focusing on the same object. In an attempt to avoid double vision, the brain will eventually disregard the image from one eye. This is referred to as suppression. In time, the ignored eye will become unable to function normally and will become largely unused. This may result in the development of lazy eye (amblyopia).

    How is strabismus diagnosed?

    Parents may be the first to notice a slight wandering of one or both of a child’s eyes. A comprehensive eye examination by a doctor of optometry is recommended by six months of age or sooner if an eye appears to be misaligned.

    How is strabismus treated?

    Treatment for strabismus can include eyeglasses (single vision or bifocal), prisms, vision therapy and in some cases, surgery. Strabismus can be corrected with excellent results if detected and treated early, which is why intervention by age six months is suggested if eyes are not aligned.


The Muromachi Period (1338-1573), also known as the Ashikaga Period, began when Ashikaga Takauji became shogun in 1338 and was characterized by chaos, violence and civil war. The Southern and Northern Courts were reunified in 1392. The period was called Muromachi for the district in which its headquarters were in Kyoto after 1378. What distinguished the Ashikaga Shogunate from that of Kamakura was that, whereas Kamakura had existed in equilibrium with the Kyoto court, Ashikaga took over the remnants of the imperial government. Nevertheless, the Ashikaga Shogunate was not as strong as the Kamakura had been and was greatly preoccupied by the civil war. Not until the rule of Ashikaga Yoshimitsu (as third shogun, 1368-94, and chancellor, 1394-1408) did a semblance of order emerge. [Source: Library of Congress]

There was almost constant warfare. Central authority had dissolved and about 20 clans fought for supremacy during a 100-year period called the “Age of the Country at War.”Ashikage Takauji, the first emperor of the Muromachi period, was regarded as a rebel against the Imperial system. Zen monks acted as advisors to shogunate and became involved in politics and political affairs. This period of Japanese history also saw the emergence of the influence of wealthy merchants who were able to create close relationships with daimyo at the expense of the samurai.

The Namboku Period (1334-1392) was relatively brief period that began with the restoration of Emperor Godaigo in 1334 after his army defeated Kamakura army during its second try. The Emperor Godaigo favored the priesthood and aristocracy at the expense of the warrior class, which rose in revolt under the leadership of Takauji Ashikaga. Ashikaga defeated Godaigo at Kyoto. He then installed a new emperor and named himself as shogun. Godaigo set up a rival court in Yoshino in 1336. The conflict between Northern Court of Ashikaga and Southern Court of Godaigo lasted for more than 60 years.

One noteworthy figure from period is Yoshimitsu, a leader who became shogun when he was 10, subdued rebellious feudal lords, helped unify southern and northern Japan, and built the Golden Temple in Kyoto. Yoshimitsu allowed the constables, who had had limited powers during the Kamakura period, to become strong regional rulers, later called daimyo (from dai, meaning great, and myoden, meanng named lands). In time, a balance of power evolved between the shogun and the daimyo; the three most prominent daimyo families rotated as deputies to the shogun at Kyoto. Yoshimitsu was finally successful in reunifying the Northern Court and the Southern Court in 1392, but, despite his promise of greater balance between the imperial lines, the Northern Court maintained control over the throne thereafter. The line of shoguns gradually weakened after Yoshimitsu and increasingly lost power to the daimyo and other regional strongmen. The shogun's decisions about imperial succession became meaningless, and the daimyo backed their own candidates. In time, the Ashikaga family had its own succession problems, resulting finally in the Onin War (1467-77), which left Kyoto devastated and effectively ended the national authority of the Shogunate. The power vacuum that ensued launched a century of anarchy. [Source: Library of Congress]

Websites and Resources

Good Websites and Sources: Essay on Kamakura and Muromachi Periods ; Middlebury College article ; Wikipedia article on the Kamakura Period Wikipedia ; Minamoto Yoritomo ; Samurai Archives article on Minamoto Yoritomo ; Wikipedia article on Muromachi Period Wikipedia ; Tale of Heike site ;

Kamakura City Websites: Kamakura Today ; Wikipedia Wikipedia ; Kamakura City site Map: Japan National Tourism Organization JNTO Kamakura Today ,aps ; Good Photos at Japan-Photo Archive

Good Japanese History Websites: History of Japan by William Gilmore-Lehne ; Wikipedia article on History of Japan Wikipedia ; Outline Chronology of Japanese Cultural History ; Samurai Archives ; National Museum of Japanese History ; Famous Personages in Japan ; Monumenta Nipponica, Respected Journal on Japanese History and Culture Links and Sources Japanese History Documentation Project ; Cambridge University Bibliography of Japanese History to 1912 ; Sengoku Daimyo ; English Translations of Important Historical Documents ; WWW-VL: History: Japan (semi good but dated source ) ; History Links ; Forums Delphi Forums, Good Discussion Group on Japanese History ; Tousando ;

Economic and Cultural Developments in the Muromachi Period

Contact with Ming Dynasty (1368-1644) China was renewed during the Muromachi period after the Chinese sought support in suppressing Japanese pirates, or wako, who controlled the seas and pillaged coastal areas of China. Wanting to improve relations with China and to rid Japan of the wako threat, Yoshimitsu accepted a relationship with the Chinese that was to last for half a century. Japanese wood, sulfur, copper ore, swords, and folding fans were traded for Chinese silk, porcelain, books, and coins, in what the Chinese considered tribute but the Japanese saw as profitable trade. [Source: Library of Congress]

During the time of the Ashikaga Shogunate, a new national culture, called Muromachi culture, emerged from the Shogunate headquarters in Kyoto to reach all levels of society. Zen Buddhism played a large role in spreading not only religious but also artistic influences, especially those derived from Chinese painting of the Chinese Song (960-1279), Yuan, and Ming dynasties. The proximity of the imperial court and the Shogunate resulted in a commingling of imperial family members, courtiers, daimyo, samurai, and Zen priests. Art of all kinds--architecture, literature, No drama, comedy, poetry, the tea ceremony, landscape gardening, and flower arranging--all flourished during Muromachi times. [Ibid]

There also was renewed interest in Shinto, which had quietly coexisted with Buddhism during the centuries of the latter's predominance. In fact, Shinto, which lacked its own scriptures and had few prayers, as a result of syncretic practices begun in the Nara period, had widely adopted Shingon Buddhist rituals. Between the eighth and fourteenth centuries, was nearly totally absorbed by Buddhism and became known as Ryobu Shinto (Dual Shinto). The Mongol invasions in the late thirteenth century, however, had evoked a national consciousness of the role of the kamikaze in defeating the enemy. Less than fifty years later (1339-43), Kitabatake Chikafusa (1293-1354), the chief commander of the Southern Court forces, wrote the Jinno sh t ki (Chronicle of the Direct Descent of the Divine Sovereigns). This chronicle emphasized the importance of maintaining the divine descent of the imperial line from Amaterasu to the current emperor, a condition that gave Japan a special national polity (kokutai). Besides reenforcing the concept of the emperor as a deity, the Jinno sh t ki provided a Shinto view of history, which stressed the divine nature of all Japanese and the country's spiritual supremacy over China and India. As a result, a change gradually occurred in the balance between the dual Buddhist-Shinto religious practice. Between the fourteenth and seventeenth centuries, Shinto reemerged as the primary belief system, developed its own philosophy and scripture (based on Confucian and Buddhist canons), and became a powerful nationalistic force. [Ibid]

Culture in the Muromachi Period

performing the tea ceremony
Under the Ashikaga shogunate, samurai warrior culture and Zen Buddhism reached its peak. Daimyos and samurai grew more powerful and promoted a martial ideology. Samurai became involved in the arts and, under the influence of Zen Buddhism, samurai artists created great works that emphasized restrain and simplicity. Landscape painting, classical noh drama, flower arranging, tea ceremony and gardening all blossomed.

Partition painting and folding screen painting were developed during Ashikaga Period (1338-1573) as a way for feudal lords to decorate their castles. This style of art featured bold India-ink lines and rich colors.

The Ashikaga Period also saw the development and popularization of hanging pictures (kakemono) and sliding panels (fusuma). These often featured images on a gilt background.

The true tea ceremony was devised by Murata Juko (died 1490), an advisor to the Shogun Ashikaga. Juko believed one of the greatest pleasures in life was to live like a hermit in harmony with nature, and he created the tea ceremony to evoke this pleasure.

The art of flower arranging developed during the Ashikaga Period along with the tea ceremony although its origins can be traced to ritual flower offerings in Buddhist temples, which began in the 6th century. Shogun Ashikaga Yoshimasa developed a sophisticated form of flower arrangement. His palaces and small tea houses contained a small alcove where a flower arrangement or work of art was placed. During this period a simple form of flower arrangement was devised for this alcove (the tokonoma) that all classes of people could enjoy.

Warfare during the period was also an inspiration for artists. Paul Theroux wrote in The Daily Beast: The Last Stand of the Kusunoki Clan, a battle fought at Shijo Nawate in 1348, is one of the enduring images in Japanese iconography, occurring in many woodblock prints (by, among others, Utagawa Kuniyoshi in the 19th century and Ogata Gekko in the early 20th), the doomed warriors defying an immense shower of arrows. These samurai who were defeated---their wounded leader committed suicide rather than be captured---are inspirational to the Japanese, representing courage and defiance, and the samurai spirit.[Source: Paul Theroux, The Daily Beast, March 20, 2011]

Warfare in the Muromachi Period

Civil wars and feudal battles occurred off and on during the unstable and chaotic 15th and 16th centuries. In the 1500s the situation got so out of out hand that bandits overthrew established leaders, and Japan almost descended into Somalia-like anarchy. During the White Sparrow Revolt in 1571 young (sparrow) monks were forced to fall to their deaths over a waterfall in the Unzen area of Kyushu.

Battles often embraced tens of thousands of samurai, supported by farmers enlisted as foot soldiers. They armies employed mass attacks with long spears. Victories were often determined by castle sieges. >Early Japanese castles were usually built on flat land in the middle of the town they protected. Later, multi-storied pagoda-like castles called donjons, were built on top of raised stone platforms.

Many important battles were fought in the mountains, difficult terrain suited for foot soldiers, not open plains where, horses and cavalries could be used to their best advantage. Fierce hand to hand battles with armor-clad Mongols showed the limitations of bows and arrows and elevated the sword and the lance as the preferred killing weapons Speed and surprise were important. Often the first group to attack the other’s encampment won.

Warfare changed when guns were introduced. "Cowardly" firearms reduced the necessity of being the strongest man. Battles became bloodier and more decisive. Not long after guns were banned warfare itself ended.

Onin War Period of Civil Wars (1467-1560)

The Onin Rebellion (Ronin Rebellion) of 1467 escalated into the 11-year Onin civil war, which was regarded as a "brush with the void." The war essentially destroyed the country. Afterwards, Japan entered the Period of Civil Wars, in which the shoguns were weak or non-existent and daimyo established fiefs as separate political entities (rather than vassals states within a shogunate) and castles were built to protect them.

The Onin War led to serious political fragmentation and obliteration of domains: a great struggle for land and power ensued among bushi chieftains until the mid-sixteenth century. Peasants rose against their landlords and samurai against their overlords as central control virtually ceased. The imperial house was left impoverished, and the Shogunate was controlled by contending chieftains in Kyoto. The provincial domains that emerged after the Onin War were smaller and easier to control. Many new small daimyo arose from among the samurai who had overthrown their great overlords. Border defenses were improved, and wellfortified castle towns were built to protect the newly opened domains, for which land surveys were made, roads built, and mines opened. New house laws provided practical means of administration, stressing duties and rules of behavior. Emphasis was put on success in war, estate management, and finance. Threatening alliances were guarded against through strict marriage rules. Aristocratic society was overwhelmingly military in character. The rest of society was controlled in a system of vassalage. The shoen were obliterated, and court nobles and absentee landlords were dispossessed. The new daimyo directly controlled the land, keeping the peasantry in permanent serfdom in exchange for protection. [Source: Library of Congress]

Most wars of the period were short and localized, although they occurred throughout Japan. By 1500 the entire country was engulfed in civil wars. Rather than disrupting the local economies, however, the frequent movement of armies stimulated the growth of transportation and communications, which in turn provided additional revenues from customs and tolls. To avoid such fees, commerce shifted to the central region, which no daimyo had been able to control, and to the Inland Sea. Economic developments and the desire to protect trade achievements brought about the establishment of merchant and artisan guilds. [Ibid]

Image Sources: 1) Samurai websites, MIT visualizing history

Text Sources: New York Times, Washington Post, Los Angeles Times, Daily Yomiuri, Times of London, Japan National Tourist Organization (JNTO), National Geographic, The New Yorker, Time, Newsweek, Reuters, AP, Lonely Planet Guides, Compton’s Encyclopedia and various books and other publications.

Page Top

© 2009 Jeffrey Hays

Last updated March 2012


Martin Scorsese

~ Person

Martin Charles Scorsese (/skɔːrˈsɛsi/; Italian: [skorˈseːze]; born November 17, 1942) is an American director, producer, screenwriter, actor, and film historian, whose career spans more than 53 years. Scorsese's body of work addresses such themes as Sicilian-American identity, Roman Catholic concepts of guilt and redemption, machismo, modern crime, and gang conflict. Many of his films are also notable for their depiction of violence and liberal use of profanity.

Part of the New Hollywood wave of filmmaking, he is widely regarded as one of the most significant and influential filmmakers in cinema history. In 1990, he founded The Film Foundation, a nonprofit organization dedicated to film preservation, and in 2007 he founded the World Cinema Foundation. He is a recipient of the AFI Life Achievement Award for his contributions to the cinema, and has won an Academy Award, a Palme d'Or, Cannes Film Festival Best Director Award, Silver Lion, Grammy Award, Emmys, Golden Globes, BAFTAs, and DGA Awards.

He has directed landmark films such as the crime film Mean Streets (1973), the vigilante-thriller Taxi Driver (1976), the biographical sports drama Raging Bull (1980), the black comedy The King of Comedy (1983), and the crime films Goodfellas (1990) and Casino (1995), all of which he collaborated on with actor and close friend Robert De Niro. Scorsese has also been noted for his collaborations with actor Leonardo DiCaprio, having directed him in five films, beginning with Gangs of New York (2002) and most recently The Wolf of Wall Street (2013).

Scorsese's other films include the concert film The Last Waltz (1978), the black comedy After Hours (1985), the epic drama The Last Temptation of Christ (1988), the psychological thrillers Cape Fear (1991) and Shutter Island (2010), the biographical drama The Aviator (2004) and the historical adventure drama Hugo (2011). His work in television includes the pilot episode of the HBO series Boardwalk Empire and Vinyl, the latter of which he also co-created. He won the Academy Award for Best Director for the crime drama The Departed (2006). With eight Best Director nominations, he is the most nominated living director, and is tied with Billy Wilder for the second most nominations overall.


Isolation and Biophysical Studies of Natural Eumelanins: Applications of Imaging Technologies and Ultrafast Spectroscopy

* Address reprint requests to Prof. John D. Simon,Department of Chemistry, Duke University, Durham, NC 27708-0346, USA. E-mail:

The major pigments found in the skin, hair, and eyes of humans and other animals are melanins. Despite significant research efforts, the current understanding of the molecular structure of melanins, the assembly of the pigment within its organelle, and the structural consequences of the association of melanins with protein and metal cations is limited. Likewise, a detailed understanding of the photochemical and photophysical properties of melanins has remained elusive. Many types of melanins have been studied to date, including natural and synthetic model pigments. Such studies are often contradictory and to some extent the diversity of systems studied may have detracted from the development of a basic understanding of the structure and function of the natural pigment. Advances in the understanding of the structure and function of melanins require careful characterization of the pigments examined so as to assure the data obtained may be relevant to the properties of the pigment in vivo. To address this issue, herein the influence of isolation procedures on the resulting structure of the pigment is examined. Sections describing the applications of new technologies to the study of melanins follow this. Advanced imaging technologies such as scanning probe microscopies are providing new insights into the morphology of the pigment assembly. Recent photochemical studies on photoreduction of cytochrome c by different mass fraction of sonicated natural melanins reveal that the photogeneration of reactive oxygen species (ROS) depends upon aggregation of melanin. Specifically, aggregation mitigates ROS photoproduction by UV-excitation, suggesting the integrity of melanosomes in tissue may play an important role in the balance between the photoprotective and photodamaging behaviors attributed to melanins. Ultrafast laser spectroscopy studies of melanins are providing insights into the time scales and mechanisms by which melanin dissipates absorbed light energy.

Abbreviations –

atomic force microscopy

5,6-dihydroxyindole carboxylic acid

minimal erythemal dose

pyrrole 3,5-dicarboxylic acid

pyrrole 2,3,5-tricarboxylic acid

reactive oxygen species

retinal pigmented epithelium

scanning electron microscopy

scanning tunneling microscopy

transmission electron microscopy

ultra-high resolution

Melanin refers to a range of biologic pigments found in a variety of locations including the hair, skin, eyes, brain, and inner ear. Melanins are commonly divided into three types: the brown-black eumelanins, the yellow-reddish pheomelanins and the dark brown neuromelanins. These three melanins are distinguished by differences in their molecular precursors (1–3). Eumelanins are composed of indolic units derived from the oxidation of tyrosine. Pheomelanins are composed of benzothiazines derived from the oxidation of cysteinyldopa units. Neuromelanins are derived from the neurotransmitter, dopamine, and have properties of both pheomelanins and eumelanins: containing both indole and benzothiazine units (4–6). In this review, we have limited the discussion to the eumelanins.

A central tenet in biochemistry is the relationship between structure and function. Given that the last few decades have witnessed significant advances in our ability to determine both the structure and function(s) of many classes of biologic molecules (e.g. proteins and nucleic acids), it is somewhat surprising that both the chemical structures and biologic role(s) of melanin are still subject to debate. To appreciate why this is the case, it is instructive to compare melanins with proteins. Features of these two classes of macromolecules are compared in Table 1 and elaborated upon below.

Table 1.  Comparison of fundamental properties of eumelanins with those of proteins
Molecular building blocksTwenty standard amino acidsDHI, DHICA are precursors to molecules present
Primary structureLinear connectivity via peptide bond, some are cross-linked by disulfide bondsUndetermined, probably involves both linear or cross-linked connectivity
3-Dimensional structuresα-Helix, β-sheet, specific folded structures based on primary sequenceUndefined higher order structure
Biogenesis mechanismWell definedMany enzymes for early steps of melanogenesis are known, but the polymerization mechanism is unknown
Synthetic preparationRecombinant and total synthetic protocols exist to make naturally occurring proteinsProtocols for making synthetic melanin, but they are structurally and chemically different than natural materials
Isolation and purificationEstablished protocols for isolation and purificationNo general methods exists, methods exist for specific tissue sources, many procedures used modify the chemical and physical properties of the pigment

1. The building blocks of proteins are the 20 standard α-amino acids. In the case of eumelanin, the basic molecular building blocks are derived from 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole carboxylic acid (DHICA). So unlike proteins, only the precursors to the molecular building blocks of melanins have been elucidated.

2. In proteins, the amino acids are connected to one another linearly through formation of peptide bond linkages. In some proteins, inter- and intra-chain disulfide bonds cross-link the polypeptide chains. In melanins, the details of the connectivity between molecular building blocks are not defined. Both linear and cross-linking connectivity have been proposed based on analysis of the early products of synthetic melanin (7, 8), X-ray scattering (9, 10), and scanning tunneling microscopy (STM) imaging (11).

3. Standard and automated methods have been developed to sequence proteins, but no method exists to determine the molecular linkage(s) of melanins. Chemical procedures can degrade proteins into their constituent amino acids, while no such chemical protocols exist to disassemble melanins into their basic molecular units. However, some chemical degradation procedures offer insights into the relative proportions of DHI and DHICA contents in the melanin pigment (12, 13). While useful and revealing, these procedures offer no information about the structure or connectivity of the molecule(s) present in the original pigment.

4. In proteins, polypeptide chains (commonly called the primary structure) adopt secondary structures (α-helix and β-sheet) and generate folded structures (tertiary structure). The three-dimensional structures of many proteins have been determined from analysis of X-ray diffraction patterns generated from a single crystal of the protein. On the contrary, in melanins, the assembly of oligomeric (or polymeric) molecules into the micrometer scale three-dimensional structures observed in vivo is not understood. A hierarchical aggregation of melanin building blocks into the three-dimensional structure has been proposed (14, 15). In this hypothesis, the fundamental units are planar highly cross-linked molecules comprised 6–8 DHI and/or DHICA units. These molecules are proposed to form layered stacks, 10–12 Å in thickness and 10–12 Å in diameter (9–11, 16). Then further lateral aggregation and stacking forms bulk melanin. X-ray scattering, STM imaging, and atomic force microscopy (AFM) imaging provides some evidence supporting this proposed aggregation structural model. Mass spectrometry studies also support the existence of small oligomers (17–19). Nuclear magnetic resonance (NMR) studies counting the number of ‘visible’ H-atoms (aromatic) in melanin also supports the proposed cross-linking between constituent DHI and/or DHICA molecules (20).

5. Standardized methods are available to synthesize proteins, both in vivo (gene recombinant techniques) and in vitro (chemical solid-state synthesis). The generated protein has the same structure and functions as the natural protein. The synthetic methods for melanins generally involve oxidation (catalyzed by enzymes or auto-oxidation) of a specific molecule (e.g. tyrosine, l-Dopa and DHI) or a small set of accepted precursor molecules. The color and solubility of the synthetic melanin mimics that of the natural melanin, however in terms of chemical composition and physical morphology, synthetic melanins are largely non-representative of natural melanins (21, 22).

6. Standardized procedures for isolation and purification of proteins from cells and tissues have been developed. Proteins are easily soluble in aqueous solution or can be solubilized using surfactants. However, melanin is essentially insoluble in any solvent. The lack of a structural knowledge about melanin at the molecular level is partially because of its insolubility and also the lack of appropriate techniques for its study.

Compared with the enormous progress in proteomics, limited progress has been made toward understanding the structure and function of melanin. Unfortunately, many of the powerful technologies used to study proteins are not amenable to the study of melanins. It is important to be aware of the above-described differences and to develop methods suitable for the study of melanin. A first step is to standardize the isolation and purification of melanin from its native environment.

As we will discuss later in this review, it is necessary to work only with well-characterized samples to reconcile the contradictory results in the current literature. These discrepancies, pertaining to the physical properties and biologic function of melanins, could be the result of many different preparative procedures used to isolate melanins, which do not share common chemical or structural characteristics.

Recent progress in the understanding of the genetic control of melanogenesis (23, 24) and the proteomics of the melanosome represent great strides forward in melanin research (25, 26). Enzymes specifically located in melanosomes catalyze many steps of the melanogenesis process. Many other proteins, non-specific to melanosomes, are also found in the organelle, and their function(s) within remain unclear, but cannot be ignored (26).

In this review article, we primarily focus on recent works towards understanding the structure and photochemistry of eumelanin. This subject matter is organized as follows. First, procedures used to isolate natural eumelanins are examined. Collectively, these studies show that the isolation procedure can modify the chemical structure of eumelanins, and so caution must be exercised in relating studies on isolated eumelanins to those in vivo. Secondly, scanning electron microscopy (SEM) and AFM studies on the surface morphology of eumelanins from human hair and the ink sac of Sepia officinalis are summarized. From a comparison of structural features of these two eumelanins, we conclude that both the natural melanin granules from Sepia and human hair and bovine eye melanosomes are aggregates of 10–30 nm substructures, consistent with the ultra-structures revealed by earlier studies of the corresponding premelanosomes. Thirdly, the effect of pigment aggregation on the aerobic photoreactivity of these eumelanins is reviewed. Fourthly, time-resolved spectroscopic studies examining the initial dynamics of the oligomeric molecules present in eumelanin from S. officinalis are discussed. These data reveal the time-scales on which eumelanin dissipates absorbed UV-radiation, and provide an explanation for the low quantum efficiency for oxygen activation observed for melanins, in general. Finally, opportunities made available by recent technical advances will be discussed as related to furthering our understanding of the structure(s) and function of melanins.

Isolation methods and comparison of the pigments they produce

Melanins are synthesized in melanosomes, a specific organelle of pigment-producing cells. Melanosomes are generated in melanocytes located at the basal layer of skin, inside hair bulbs, and in some tissues of the eye. Melanosomes can also be found in the postmitotic cells of the retinal pigment epithelium (RPE) in eyes (27–29). Melanosomes can be delivered to other cells, as evidenced by the transfer of dermal melanosomes from melanocytes to neighboring keratinocytes. In some species, melanosomes are released into an extracellular compartment, e.g. the pigment-producing epithelial cells lining the ink sac of cuttlefish release the melanosomes into the lumen of the ink sac (30).

Melanin in vivo is always associated with proteins, which were present in the premelanosomes. Some of the proteinaceous material present is likely to be the remnants of the enzymes that catalyzed various steps of melanogenesis (31). In the 1960s, several research groups used transmission electron microscopy (TEM) to examine the ultrastructure of premelanosomes (32–36). These studies revealed filaments and sheets-like structures inside the premelanosomes at early stages of melanogenesis. This material was shown not to be lipid membrane and was proposed to serve to localize and control melanin deposition within the organelle. Evidence has suggested this material is protein (35), but its exact structure and composition is not yet known. To study melanin requires separation and purification of the pigment from its biologic environment. The challenge is to develop procedures that do not modify the pigment during isolation, so the pigment obtained is reflective of its native form. Melanins found in different tissues require different isolation procedures. The strengths and weaknesses of various approaches are described below.

Sepia Officinalis

We first consider melanin from the ink sacs of cuttlefish such as S. officinalis. Melanogenesis in the ink gland of S. officinalis was recently reviewed (30). Sepia eumelanin is considered to be pure eumelanin and is used as a standard for natural eumelanins (37, 38). Sepia eumelanin is isolated through iterative washings of the ink with water and centrifugation. The pellet obtained after extensive washings contains largely spherical granules ∼150 nm in diameter (39). This shape and size is consistent with TEM images of granule production within the Sepia melanosome (40). SEM and AFM images of isolated granules are shown in Figs 1 and 2, respectively. Chemical analysis reveals these granules have a protein content of 6–8% by mass (39, 41, 42). In general, no attempt was made to separate melanin from these internal proteins, because such a separation would destroy the granules.

Figure 1.

In the isolation of Sepia eumelanin from its ink sac, different drying processes result in the formation of different aggregated structures. Under high magnification, all aggregates are composed of spherical granule of diameter ∼150 nm, which is the natural morphology. (A) Spray drying prepared by Mel-Co (Whittier, CA) and Sigma (St. Louis, MO) . Air-drying (B), freeze-drying (C), and CO2-supercritical drying (D) prepared from fresh ink sacs. The scale bars are 20 μm on the low magnification images (left) and 2 μm on the higher magnification images (right). Some of the images are reprinted with permission from Liu and Simon (39).

Figure 2.

AFM images of Sepia granules deposited on mica. (A) Height image, (B) phase image. Scan size is 383 × 383 nm. These images reveal the 10–30 nm substructures that comprise the granule.

This isolation procedure serves to illustrate the first caution in melanin research. Much of what we know about the chemical and physical properties of melanins has been determined on synthetic pigments. Such ‘melanins’ may not contain any protein and certainly do not contain all the proteins present in melanosomes. In contrast, natural pigments contain both melanin (the organic component derived from the initial oxidation of tyrosine) and protein. As described above, the protein component is important in defining the assembly of melanin, and it is reasonable to assume through this mechanism the protein that affects the function of the assembled pigment. Furthermore, in the study of natural pigments reported in the literature, the term ‘melanin’ is used loosely and can represent materials with various protein contents.

Caution should not be only limited to the association of proteins with melanin. A second caution concerns the variety of metal cations present in the natural pigment and absent in the synthetic pigments unless added in the reaction mixture. Even if added to the synthetic systems, the coordination of the metal ions in vivo is likely different from that in vitro. The concentration of these metals also varies among natural melanins (43–45), although it is unclear how the isolation procedure affects these results. It is also possible the metals play a significant functional role in the pigment, which would not be manifested in studies of synthetic systems.

A third caution highlighted in the analysis of isolated Sepia granules is that the drying method employed yields different aggregated structures (Fig. 1). The method chosen affects the physical properties of the pigment, e.g. surface area to mass ratio and porosity of the material (39). The drying process does not affect the chemical composition. However, as will be discussed in the following section, the chemical reactivity of the pigment is dependent on aggregation (46). Therefore, care should be taken when comparing the photobiologic data obtained from samples using different preparation procedures.

Sucrose Gradient Purification

In some cases, pigmented cells can be lysed and intact melanosomes can be isolated from other cellular constituents, e.g. RPE and melanoma cells. Ultracentrifugation on a discontinuous sucrose gradient (47) yields high purity samples where the integrity of the melanosomes is preserved. This is exemplified by the AFM images of bovine RPE melanosomes (Fig. 3) isolated in this manner.

Figure 3.

AFM images of bovine eye melanosomes on mica. The melanosomes were isolated from bovine RPE cell lysate using a discontinuous sucrose gradient. The melanosomes were washed with distilled water prior to imaging. (A) Scan size is 3.08 × 3.08 μm. (B) Higher resolution scan of a region shown in (A); scan size 800 × 800 nm. Left panel is height image, and the right panel is phase image. Similar to Sepia, these images reveal the melanosomes is an aggregated assembly of ∼20 nm substructures.

Melanosomes in Complex Environments

Generally, melanosomes are constrained in a biologically complex environment, e.g. in hair or the brain, and their isolation is often a significant challenge. The separation of melanin from such tissues has largely been achieved by using harsh protocols. These protocols often involve repeated treatments with concentrated acid and base breaking down the surrounding tissue to separate the melanin from proteins and other biologic materials. In many such studies melanin was assumed to be resistant to degradation by the chemicals used in the isolation. As hair is the only readily available non-invasive human sample, pigments isolated from hair have been widely used as model for natural human melanin. In hair, the melanin is tightly held within a keratin matrix. Most studies of hair melanins degrade this protein matrix by exposing the hair to harsh chemical environments such as strong alkali (48), hydrazine/ethanol mixtures (49), or thioglycolic acid/phenol mixtures (50). The acid hydrolytic methods induce the protein to form pigment-like artifacts interfering with the isolation of natural melanin pigments, whereas alkaline hydrolytic methods cause transformation of the chemical composition of melanin pigment and associated protein (51). In particular, it has been reported that exposure to acid results in extensive decarboxylation of eumelanins (22). Electron microscopy imaging demonstrated acid/base treatments modify and disrupt the ultrastructure of melanosomes (51, 52). This necessitated changing the approach for degrading the keratin matrix that would leave the hair melanosome intact and chemically unaltered.

A major advance was made in 1956 when Birbeck et al. used urea, papain and sodium metabisulfide to isolate melanosomes from human hair (53). They found the hair melanin granules isolated by enzymatic digestion were more homogenous than both acid and base extracted melanin. In 1973, Bratosin reported a method for isolation of melanosomes from black mouse hair (54). Keratin was removed from the hair through a two-step process consisting of alkaline hydrolysis and subsequent enzymatic (trypsin) digestion. It was observed that the isolated melanosomes retain their intact morphologic structure. In 1981, Arnaud and Bore used proteinase PSF 2019 to isolate human hair melanin (51). Incubation in either DMF or concentrated LiBr was required to accelerate the subsequent enzymatic digestion. The enzymatic digestion went on for 8 d at pH 9. Afterward, the hair was treated in three acid hydrolysis steps to remove external proteins associated with melanin.

All three of these enzymatic methods were used in conjunction with either alkaline hydrolysis or acid hydrolysis, the drawbacks of which drew attention in the mid 1980s (22, 52). In the 1990s, new procedures for enzymatic isolation were developed, mostly in response to desires to study neuromelanin. In the isolation of neuromelanin from the substantia nigra of human brain, proteinase K (55–57), collagenase (6), and pronase E (58) were used.

In 2000, Prota and co-workers introduced another enzymatic procedure for the isolation of melanins from human hair and iris (19). Using proteinase K, papain and protease type XIV consecutively and a detergent Triton-X-100, this method is truly proteolytic and without any harsh chemical reagents. These workers found the chemical and spectral characterization of the pigments from human hair and iris revealed marked structural differences, which challenged the common belief that all melanins are similar independent of their origin. They further suggested this could be due to the diversity of functions and sites of biosynthesis and storage. This conclusion is based on the assumption that the enzymatic isolation procedure produces a pigment representative of melanin in its native environment.

In a recent study (43), we examined eumelanin from a sample of black, Indonesian hair using three different published procedures: two acid/base extractions (59, 60) and an enzymatic extraction (19). The morphology and spectroscopic properties of the isolated pigments differ significantly. The acid/base procedures both yield an amorphous material, whereas enzymatic extraction yields ellipsoidal melanosomes (Fig. 4). Amino acid analysis shows there is still a significant amount of protein associated with the isolated pigments, counting for 52, 40 and 14% of the total mass for the two acid/base extractions and the enzymatic extraction, respectively. Neither of the amino acid compositions correlate with keratin or tyrosinase. Metal elemental analysis by inductively coupled plasma mass spectrometry shows that the acid/base extraction removes the majority of many metal ions bound to the pigment. Chemical degradation analysis by KMnO4 and H2O2/OH indicates significant differences between the pigments by the acid/base extractions and the enzymatic extraction. After correction of the protein mass in the two pigments, the factor of 2–3 lower yields of both pyrrole 2,3,5-tricarboxylic acid (PTCA) and pyrrole 3,5-dicarboxylic acid (PDCA) indicates 45–65% of acid/base-extracted melanin has been chemically modified, consistent with the result of the Soluene solubilization assay. While the optical absorption spectra of the bulk pigments are similar, the spectra of the molecular weight <1000 amu fractions differ significantly. The data clearly indicate pigment obtained from human hair by acid/base extraction does not effectively separate the pigment from amino acids, and the conditions lead to the destruction of the melanosome and alter the molecular structure of melanin. The acid/base extracted hair melanin is not representative of the natural material and is a poor model system for studying the physical and biologic properties of melanins. The enzymatic extraction preserves the integrity of the melanosome, removes most of the external proteins, and therefore should be the preferred choice for isolation of melanin from hair samples. At present, the procedure reported by Prota and co-workers (19) is the best approach for isolating melanosomes from hair and iris samples.

Figure 4.

SEM images of hair eumelanin obtained using different extraction methods. The detailed procedures can be found in (43). (A) and (B) show the results from two different acid/base procedures. (C) shows the results from an enzymatic method. Only the enzymatic method preserves the natural morphology of the melanosome, and is clearly the method of choice for isolating hair eumelanin for physical and photobiological studies. All images are reprinted with permission from Liu et al. (43).

Morphology of melanin assemblies as determined by modern imaging techniques

A variety of mass spectrometry and imaging studies on synthetic eumelanins argue that the pigment is an aggregated system of small oligomeric (∼4–6 monomers) molecules. Supporting mass spectrometry data have been reported for synthetic (61–63) and natural melanins, such as isolated from Sepia (64), human hair (65) and iris (19). Solubilization of natural pigment by detergent solution suggested the dominant forces holding the melanin granules together are not covalent in nature, but rather arise from hydrophobic interactions (54). However, to date the relative contributions of Van der Waal's forces and hydrogen bonding are not clear.

AFM is an imaging technique that provides three-dimensional topographical information. The AFM tip is located at the end of an oscillating cantilever. In tapping mode, this tip is scanned in a two dimensional raster pattern over the sample, and the height is adjusted to maintain the amplitude of the oscillation. In general, the height and lateral resolution of the image are 0.1 and ≤10 nm (depending of the dimensions of the scanning tip), respectively. Phase images can be collected simultaneously with the height image. Phase data measure the phase shift in the cantilever oscillation from that of the driving force. This results from attractive and repulsive interactions between the AFM tip and the sample. The phase signal can be related to the stiffness of the sample and is useful for revealing domains that may otherwise be hidden, overlooked, or difficult to see in the height image.

In a recent AFM study on mass-selected fractions of Sepia eumelanin, many of the observations reported were consistent with the conclusion that the pigment is derived from small oligomeric species (15). First, in experiments utilizing the AFM tip to cut across granules, images revealed that the tip could be used to cut ∼30 nm deep and ∼30 nm wide troughs through a granule. Accompanying the cut, a ∼20-nm ridge is generated, revealing the material displaced by the act of cutting (Fig. 5). These data indicate that the tip displaces constituents that are small compared with its dimensions. Secondly, to gain more information on the dimensions of these molecular components, images were collected on a dried 1000 < MW < 3000 sample. For that sample, significant regions of the mica are covered with a filament structure (Fig. 6). The filaments are 3–6 nm in height, 15–50 nm in width, and often several microns in length. They are easily cut along the 15–50 nm axis using the AFM tip. The data support the hypothesis that these are stacked structures, not extended covalently bound structures. This result is consistent with the mass spectrometry and X-ray scattering studies suggesting the building blocks of the pigments are small oligomeric molecules. These studies also clearly reveal the granules are aggregated structures. When we consider the 3000 < MW < 10000 fraction, height images of this sample dried on mica reveal fractal-like growth patterns (Fig. 7). The fractal aggregation is likely a result of drying a hydrophobic material at low concentration on a hydrophilic surface. The growth involves the re-aggregation of small structures, but we do not observe any evidence for the reassembly of the material into granules. This suggests the granules cannot self-assemble from the constituent oligomers under the conditions used in vitro.

Figure 5.

(A) Tapping mode AFM height images of a Sepia melanin aggregate before (left) and after (right) the AFM tip was used to cut across the aggregate along the direction indicated by the white arrow. The scale bar is 210 nm. (B) Cross-sections along the solid line (perpendicular to the cut) shown in (A) before and after cutting the aggregate. The images are reprinted with permission from Clancy and Simon (15). These images show that the act of cutting displaces molecular constituents that are small compared with the dimensions of the AFM tip.

Figure 6.

Tapping mode AFM phase image of eumelanin filaments formed from the 1000 < MW < 3000 Sepia eumelanin fraction upon drying on mica. The scale bar is 250 nm. The filaments are 3–6 nm in height, 15–50 nm in width, and often several microns in length. This further supports that natural eumelanin structures are not large cross-linked polymers but are aggregates of molecular oligomers, whose structure(s) are yet to be determined. The image is reprinted with permission from Clancy and Simon (15).

Figure 7.

AFM image of mass fraction of 3000 < MW < 10000 of Sigma Sepia melanin deposited on mica. (A) Height image, scan size is 19.6 × 19.6 μm. (B) Height image, scan area is 7.7 × 7.7 μm. (C) Height image, scan size is 2.65 × 2.65 μm. (D) Phase image, scan size is 310 × 310 nm. A series of images of different scan scales are shown to illustrate the fractal pattern, which likely results from drying a hydrophobic material (eumelanin) at low concentration on a hydrophilic surface (mica). The deposit reveals reaggregation of small structures, but the reassembly of this material into its natural morphology (granules) has not been achieved in vitro. The images are reprinted with permission from Liu and Simon (39).

While the complete details of the assembly of the granules in vivo remains unknown, additional insight into the substructure can be obtained from high-resolution SEM and AFM studies (39). Fig. 8A shows an ultra-high resolution (UHR)–SEM image of granules deposited on mica. Two important features are manifested in this image. First, the surface of the granules appears to exhibit some substructures, with constituents being ∼10–20 nm in lateral dimension. Secondly, in regions where granules are in contact, the shape of a granule changes from spherical to hexagonal. This not only represents optimal two-dimensional packing, but also clearly indicates that the granules have a degree of plasticity and can be somewhat deformed without disintegration. The preparation of the sample imaged in Fig. 8A involves coating the materials with an Au/Pd mist. This could give rise to the deposition of Au/Pd colloidal particles on the surface, which could mistakenly be interpreted as substructures. To address this possibility UHR–SEM images of uncoated melanin, deposited on conductive substrate, such as silicon wafer, were examined. The quality of the images is dramatically reduced but one can still discern the roughness of the surfaces (Fig. 8B). Pigment granules without any coating can also be imaged by tapping mode AFM, the variation of the height of the cantilever across the scanning area (height image) gives real three-dimensional topology information and the variation of the phase of the cantilever vibration across the scanning area (phase image) can provide information concerning if boundaries exist between regions of differing hardness. Thus, verification of substructure revealed by the SEM images of coated samples is possible by examining AFM images of uncoated granules, as shown in Fig. 2A,B. The images clearly show similar substructure for the granules. These results suggest that the granules are aggregates of ∼10 nm constituents. UHR–SEM images of sonicated Sepia samples subjected to five cycles of sonication and centrifugation also support the aggregation model (Fig. 9). Thus sonication in a water bath introduces mechanical perturbation strong enough to remove the ∼10 nm constituents from the surface of the granules or cause a partial decomposition of the granules.

Figure 8.

(A) UHR–SEM images of granules from the Sepia ink sac deposited on mica. The sample has been coated with colloidal Au/Pd. (B) UHR–SEM image of uncoated granules deposited on silicon wafer. Comparison of the images of the coated and uncoated samples show the surface substructure is inherent to the melanin and not the result of the protocol used to collect the images. The images are reprinted with permission from Liu and Simon (39).

Figure 9.

UHR–SEM images of supernatant after fifth round sonication and centrifugation, air-dried on mica. The images clearly show sonication can result in deformation of the granules. In addition, a thin layer of material coats some of the granules, which is not melanin, but a remnant of other cellular debris present in the ink sac. This material can be remove from the melanin following more extensive washing. The images are reprinted with permission from Liu and Simon (39).

It is interesting to compare the AFM images of melanin from the Sepia ink sac (Fig. 2) with that of the human hair melanosome isolated enzymatically (Fig. 10). Although the two natural melanin granules have different shapes, spherical vs. ellipsoidal, and different dimensions, roughly 150 nm vs. 450 nm × 1 μm, the surface features look the same. The surfaces of both granules are clearly not smooth but with substructures in the size range of a few nanometer to 30 nm. The similarity between human hair melanosomes with bovine eye melanosome is more striking (Fig. 3). Although they are also of different size (bovine eye melanosomes are ∼900 nm × 2 μm), the shape is similar and the surface features are essentially indistinguishable. Amino acid analysis on the natural melanin indicates there is a significant amount of protein associated with melanin (8% in sepia and 14% in hair by mass). Based on mass, if we approximate the eumelanin oligomer(s) as 1000 amu, then a typical melanosome represents an assembly of more than a million such building blocks. The well-defined structure revealed by imaging experiments strongly suggests that the assembly of the fully pigmented melanosome is a tightly controlled biologic process. Thus, not only are the primary and early oxidation steps of melanogenesis under enzymatic control, but also the assembly of the deposited pigment within the melanosomes must also be controlled, possibly by distribution and localization of the enzymes in the internal membrane structure of the premelanosomes (66, 67).

Figure 10.

AFM images of a single human hair melanosome. The scan size is 1 × 1 μm. Left panel is height image, and the right panel is phase image. Similar to Sepia and bovine eye melanosomes (Figs 2 and 3) these images reveal the hair melanosomes is an aggregated assembly of ∼20 nm substructures.

Aggregation affects photogeneration of reactive oxygen species (ROS)

Photochemical excitation of melanin results in the activation of oxygen, the most important primary photochemical pathway being the formation of the superoxide anion, Oinline image. In an effort to quantify the activation of molecular oxygen by eumelanin, Sarna and co-workers determined the action spectrum for the photoconsumption of oxygen by synthetic and natural eumelanins (68). While eumelanin exhibit absorption throughout the visible and ultraviolet region, oxygen photoconsumption occurs only for wavelengths shorter than 400 nm. In their original report Sarna and co-workers suggested that the chromophore responsible for oxygen photoconsumption differed from that which dominated the absorption spectrum. One explanation would be that eumelanin is composed of a number of molecular entities and one particular constituent has an absorption spectrum matching the action spectrum. Given the above discussion of the structural morphology of the pigment, however, it is reasonable to propose that different sized aggregates have different spectroscopic and photoreactive properties. In this case, a specific arrangement of the oligomeric building blocks (individual or aggregated) would be responsible for the photoconsumption of oxygen.

Different mass-selected fractions of eumelanin solutions were obtained by ultrafiltration, the optical properties of which show that the absorption for the MW < 1000 fraction of eumelanin from both Sepia (69) and black human hair (70) match the action spectrum for photoconsumption of oxygen (Fig. 11). Samples of larger masses (e.g. 1000 < MW < 3000, MW > 10 000) exhibit increased absorption at longer wavelengths, and whether these fractions are aggregates of the oligomers present in the MW < 1000 solution or longer oligomers remains to be determined. Photoacoustic calorimetry revealed the MW < 1000 sample is the only mass fraction showing measurable energy storage following UV-A excitation (71). These data suggest unaggregated oligomers underlie the phototoxic effects of melanin and aggregation mitigates such processes.

Figure 11.

The optical spectra for MW < 1000 fractions of (- - -) hair eumelanin and (…) Sepia eumelanin with the 270-nm absorption feature removed are compared with (-) the action spectrum for the free-radical photogeneration by eumelanin. Quantitative agreement is observed. These data suggests the unaggregated oligomers make the dominant contribution to the photoconsumption of oxygen by eumelanins. The similarity observed among Sepia and human hair suggests that they contain similar if not identical molecular species. The action spectrum is reproduced with permission from Sarna and Sealy (68).

It is important to ask how and if aggregation affects the mechanism and yield of ROS photoproduction by eumelanin. To address this issue, the reduction of cytochrome c (cyt c) by eumelanin excited by UV-B (302 nm) was examined. The following conclusions could be drawn from that study (46).

First, while the initial reduction rate of cyt c does not change with aggregation, the optical density of the solution does. As a result the apparent quantum yield for Oinline image formation decreases with aggregation.

Secondly, studies with biochemical quenchers reveal a quantum efficiency of hydrogen peroxide, H2O2, production by the MW < 1000 fraction of 5.7 × 10−3. The formation of H2O2 is attributed to reaction between Oinline image and semihydroquinone groups on the oligomeric molecules. The quantum yield becomes immeasurable upon aggregation of the oligomers and the mechanisms of this quenching remains to be established. In any event, aggregation clearly reduces the production of this highly toxic oxidant.

The aggregation-dependent generation of ROS by eumelanin presents a framework for understanding contrasting photoprotective and phototoxic roles exhibited by eumelanin. The equivalence between the action spectrum and absorption spectrum along with the aggregation-dependent quantum efficiencies for Oinline image and H2O2 implicate the oligomers as the phototoxic component. Thus, any changes in melanin that lead to a disruption of the aggregated structure could result in increased oxidative stress. The fact that oligomers generate more H2O2 than aggregated pigment may have significant biologic ramifications. H2O2 can react with a variety of cellular components, causing, for example, lipid peroxidation of membrane and hydroxylation of proteins and DNA. Such processes may be important in skin keratinocytes, where melanosomes are partially degraded (72), or in retinal pigment epithelium cells where the structural features of melanosomes are found to change with age (73, 74).

Probing non-radiative relaxation of eumelanins by time-resolved optical spectroscopy

The low emission quantum yield (10−5–10−3 depending on the aggregation) by photoexcited eumelanin suggests the pigment efficiently releases the absorbed light energy through non-radiative means (75). Furthermore, the low quantum yield for oxygen activation (10−4–10−3 depending on the aggregation) suggests that the dominant process is likely rapid relaxation to the ground electronic state of the pigment (46). Such a conclusion is supported by photoacoustic studies of melanins (71), but the time resolution of this technique only reveals that non-radiative relaxation occurs on the sub-nanosecond time scale.

The initial photophysical and photochemical events following UV excitation of melanins can be quantified by using time-resolved optical spectroscopy. Both time-resolved absorption and fluorescence experiments have been performed on melanins. Time resolved absorption studies suggest rapid repopulation of the ground state following UV-A and UV-B excitation (T. Ye and J.D. Simon, unpublished data). The recovery dynamics are complex and three time constants are revealed (Table 2). Despite the complexity of the decay dynamics, it is clear that melanin efficiently and rapidly converts absorbed photon energy into heat. The rapidity of this process mitigates the potential of adverse photochemistry and supports the hypothesis that melanin exhibits, in part, a role in photoprotection.

Table 2.  Time constants and amplitudes (in parentheses) from time-resolved experiments on eumelanin
  1. *The amplitudes of these decay constants vary with probe wavelength.

Transient absorption*0.56 ± 0.8 ps3.2 ± 0.5 ps31 ± 5 ps
Emission decay58 ± 7 ps (0.54)0.51 ± 0.07 ns (0.22)2.9 ± 0.5 ns (0.16)7 ± 1 ns (0.08)

The decay associated spectrum for each time constant could provide information of the spectral regions associated with a particular decay component. In this manner, it may be possible to decompose the transient spectrum into contributions for different molecular entities, each of which exhibits a particular decay time constant. Unfortunately, in the case of eumelanin, such an analysis does not cleanly assign a specific absorption feature in the transient spectrum to a given time constant. The resolution afforded by the global data analysis suggests that all spectral features present in the transient spectrum contribute to the multi-exponential decay. It is likely that these spectra are derived from a small set of structurally similar chromophores within the eumelanin pigment. This would explain the non-exponentiality of the decays, the similar vibronic structure observed in the transient spectrum, and the differences in the relative intensities of the vibronic bands. Given that the connectivity of the monomer units in the structure of eumelanin is not known, it is currently not possible to speculate on the molecular details that give rise to these spectral differences.

It is interesting to compare these dynamics with those obtained from fluorescence lifetime measurements. The emission dynamics of melanin are non-exponential and require a sum of exponentials to generate functional forms that provide fits to experimental data. In a previous report on Sepia eumelanin (76), four time constants were needed to describe the emission collected at 520 nm (near the maximum of the spectrum) following excitation at 355 nm (see Table 2). It is interesting to note that the fastest lifetime component of the emission is in rough agreement with the slowest time constant revealed by the ultrafast absorption measurements, and thus it is possible that both experimental techniques ‘sense’ the decay of the same molecular species, but the time resolution of the emission experiments is not sufficient to observe the faster decay component.

One must exercise caution in interpreting the transient spectroscopic results of melanins in terms of the photobiologic properties of the pigments. While interesting dynamics are revealed, it remains to be established whether either transient absorption or emission experiments probe the molecules responsible for the photoaerobic reactivity of melanins. To address such issues, it will be important to measure the transient optical properties and the action spectra for photoaerobic processes (oxygen photoconsumption, superoxide formation) on the same set of samples. Comparison between existing published literatures is potentially problematic resulting from the range of methods used to isolate and prepare melanins and the effects these procedures have on the integrity and molecular structure of the pigment.

Future directions

This is an exciting time in melanin research. The last decade has witnessed the development of a diverse set of powerful analytical tools, many of which can be applied to characterize natural pigments. Advances in biochemical techniques are now enabling the isolation of fully assembled melanosomes, and so one can start asking detailed biophysical questions concerning the nature of the assembly. We are optimistic that significant advances will be forthcoming over the next several years. The following topics are presented in a hope to excite and inspire those either working in the field or interested in contributing to biophysical studies of melanins.

Molecular Structure(s) of the Oligomers

While the early steps of melanogenesis are understood (77–80), the molecular structures of the resulting oligomers remain elusive. Mass spectral studies have provided some insights into the molecular weights of constituent oligomers, and oligomers of DHI and/or DHICA with a linear connectivity have been proposed (19, 62–64). Recent NMR studies of melanins suggest cross-linked connectivity between monomers (20). Highly cross-linked planar oligomeric structures have been suggested by X-ray scattering (10, 81) and STM experiments (11, 82). At this point of time, it is unclear whether melanogenesis produces a heterogeneous set of oligomers of appreciable relative concentrations or a single molecular species dominates. Recent advances in high-performance liquid chromatography (HPLC)–MS/MS techniques suggest that this analytical tool could play a powerful role in elucidating the structures of oligomers. While standard MS techniques provide the molecular weight, MS/MS and higher order fragmentation can provide information on the detailed structure. In addition, the recent commercialization of HPLC–NMR may also prove to be a valuable technique for structural determination.

The molecular structure(s) of melanin is one area where theoretical insights currently outpace experimental achievements. Galvao and Caldas were the first to carry out theoretical calculations on model polymers for eumelanins in late 1980s (83, 84). In the last 2 yr, several groups used modern computational chemistry tools to examine the structure and spectroscopy of monomers (85), dimers (86, 87), and oligomers of eumelanin (88, 89). While these papers are of interest in their own right, there is no evidence that the molecular structures explored are actually present in melanin. Experiments are surely needed in this area. With knowledge of naturally occurring structures, the computational tools could be used to provide a deep understanding of the spectroscopy and association of these molecules in the aggregated pigment.

This article has focused on eumelanin. In nature there is a second pigment, pheomelanin, which is yellow-reddish in color. The biogenesis of pheomelanin differs from eumelanin in that cysteine is incorporated into the structure and the building blocks are derived from cysteinyl-dopa. Epidemiological data indicate individuals with fair skin are more susceptible to skin cancers than their darker skin counterparts (3). This observation is commonly associated with the hypothesis that pheomelanin exhibits a greater phototoxicity than eumelanin. In support of this hypothesis, Prota and co-workers explored whether there was a relationship between hair melanin composition and minimal erythemal dose (MED) in a group of red-haired individuals (90). They found a correlation between the eumelanin/pheomelanin ratio and the MED values, suggesting UV sensitivity is associated with high pheomelanin and low eumelanin levels and that the eumelanin/pheomelanin ratio may be a chemical parameter for predicting individuals at high risk for skin cancer and melanoma. Cellular studies support this general concept; UV-A-induced DNA single-strand breaks in human melanocytes differing only in the amount of pigment produced showed photosensitization by intrinsic chromophores, most likely pheomelanin and/or melanin intermediates (91). These results indicate the need to understand the molecular composition, the structure, and the photobiology of pheomelanin. To date, most of our limited knowledge on this pigment comes from the study of synthetic samples. Careful studies on pheomelanin from natural systems are needed, as are quantitative comparisons between such a sample and a related natural eumelanin.

The Role of Metals

Melanin is able to sequester heavy metals ions, such as iron, copper, zinc and lead, in principle protecting the surrounding tissue from their cytotoxicity. However, it is also proposed that when the binding sites of a melanosome become saturated, the integrity of melanosome could be compromised, and heavy metal ions and/or melanin oligomers could be released from melanin and trigger acute damage to the cell (92). Along these lines, a link between Fe(III) binding to neuromelanin and death of pigmented neurons and pathogenesis of Parkinson's disease has been proposed (93, 94).

To what extent melanin can bind metals like Fe(III) and how this binding affects the structure and function of the pigment represent a set of interesting questions, which have not received careful attention. There is clearly an opportunity to carry out significant work in this area, especially if the work can be carried out with well-characterized samples of neuromelanin.

Molecular Aspects of the Aging Melanosome

Sarna and co-workers examined the photochemically induced uptake of oxygen by different age cohorts of human RPE melanosomes (95). These data clearly show the increase of the activation of oxygen by melanosomes with increasing age (Fig. 12). The emission intensity of RPE melanosomes also increases with age (96). It is interesting to note that Schraermeyer and co-workers recently demonstrated that melanin fluorescence from synthetic melanin and melanin isolated from bovine melanosomes increases after oxidation (97). One could therefore hypothesize that the age-dependent aerobic photoreactivity of RPE melanosomes reflect concomitant changes in the molecular structure of the melanin (e.g. oxidative damage) and/or increased concentrations of bound redox-active metal cations. Because of the potential link between changes in the photobiology of retinal melanosomes and cell atrophy of the RPE layer, a molecular understanding of the origin of these effects would be important in the prevention of diseases in this tissue.

Figure 12.

Broadband, blue-light-induced oxygen uptake in suspensions of melanosomes isolated from donors in the following ages: <40 yr (□), 42–60 yr (▵), 61–80 yr (○), >80 yr (◊). Oxygen uptake in the dark was negligible in all samples studied. The concentration of pigment granules was adjusted to 4 × 109 granules/ml. Oxygen uptake was measured with ESR oximetry. The data clearly show the aerobic photoreactivity of melanosomes increases with age. The images are reprinted with permission from Rozanowska et al. (95).

Acknowledgements– This work was partially supported by the National Institute of General Medical Sciences. We thank Unilever Research US for continued support of this work. We also thank the following co-workers for their contributions to the work described herein: Dr Susan E. Forest, Dr Chris M.R. Clancy, Dr J. Brian Nofsinger, Dr Tong Ye, Valerie R. Kempf, Prof. Shosuke Ito, Prof. Kazumasa Wakamatsu, Dr Mark Rudnicki, Emily E. Weinert, Leslie Eibest, Prof. Yuri Il'ichev, and Prof. T. Sarna.


AUSTIN, Texas—An Austin, Texas, technology company says 20 of its employees were aboard the Malaysia Airlines plane that went missing over the South China Sea.

Jacey Zuniga, a spokeswoman for Freescale Semiconductor, says 12 Malaysian and 8 Chinese employees are "confirmed passengers." She says no American citizen Freescale employees were on the flight.

"At present, we are solely focused on our employees and their families," Gregg Lowe, president and CEO of Freescale says in a statement. "Our thoughts and prayers are with those affected by this tragic event." The company, the statement reads, has assembled a team of counselors for those impacted by the tragedy.

Flight MH370, a Boeing 777 airplane, was last seen on radar at 1:30 a.m. (1730 GMT Friday) above the waters where the South China sea.

Freescale Semiconductor is a technology company focused on what it calls "embedded processing solutions." It works with clients in a variety of markets, including automotive and consumer electronics, to address technology issues using microprocessors and sensors.


Placement of Fingers in Icons

What is the significance of the placement of the fingers of the right hand I notice in icons of Christ and certain saints?

The fingers are arranged to form the following letters—IC XC—which are the first and last letters of “Jesus” and “Christ” in Greek.