We will be returning to 1 John today where John has given us so much truth, foundational doctrine, and hope. He has also given us tests to determine true Christians, those who claim to be Christians. But, today, John is going to remind us that it is all about Jesus Christ. He is going to highlight three different aspects of God the Father's witness of Jesus Christ His Son. John will also reveal the purpose of that testimony. And finally, John illustrates the power of that testimony. In 1 John 5:6-12, John says….
Throughout this letter, John has consistently and persistently hammered home this truth; a correct view of the Lord Jesus Christ is essential to salvation. This has been John's theme from the very beginning of this letter. Just look at how John starts the letter in verses 1 – 4.
And finally in verse 20, John writes.
The Old Testament predicted the precise details of Jesus' life. Isaiah prophesied that He would be born of a virgin (7:14). Micah prophesied that He would be born in Bethlehem (5:2). Hosea prophesied that He would be called out of Egypt (11:1). Malachi prophesied the forerunner (John the Baptist) who would prepare the way for Him (4:5–6). Psalm 41:9 predicts His betrayal by a close friend, Psalm 22 delineates the details of His crucifixion, Isaiah 53 explains the theological significance of His death, and Psalm 16 foresees His resurrection.
O foolish men and slow of heart to believe in all that the prophets have spoken! Was it not necessary for the Christ to suffer these things and to enter into His glory?
These are My words which I spoke to you while I was still with you, that all things which are written about Me in the Law of Moses and the Prophets and the Psalms must be fulfilled.
written so that [people] may believe that Jesus is the Christ, the Son of God; and that believing [they] may have life in His name (John 20:31).
However, the key to this section is the word "testify", which appears (in both noun and verb form) nine times in verses 6–12. The root word however is a common word that appears nearly 175 times in its various forms in the New Testament. It has the basic meaning of remembering something and testifying concerning it. That testimony could be either in a legal setting or in the general sense of recounting firsthand knowledge.
In the Old Testament, God called the people of Israel to be witnesses that He alone is God and to testify of this truth. But, the New Testament is supremely God's witness to His Son. The Gospels record the story of Christ's life, death, and resurrection; the book of Acts describes the initial spread of the truth about Him into the world; the epistles unpack the rich theological meaning of His life, death, resurrection, ascension, and second coming; the book of Revelation reveals the consummation of God's redemptive purpose in Him.
The Jews scornfully referred to Him as a lying deceiver, guilty of leading the people astray and of provoking an insurrection against Rome. They even blasphemously accused Him of being a glutton and a drunkard, of being insane, and, most heinous of all, of being demon-possessed. Because of their murderous hatred of Jesus, they called for His crucifixion. In light of Israel's rejection, why should anyone believe that Jesus Christ is the Messiah, God incarnate in human flesh, the only Savior of sinners? John tells us why! Because of the infallible, unquestionable, incontrovertible testimony of God Himself.
John did not say Jesus was created, but instead, he said, Jesus Christ appeared.
There are various attempts to connect the phrase "water and blood" with Jesus' death, when one of the soldiers pierced His side with a spear, and immediately blood and water came out. But there is no reason to assume that John had that incident in mind. Others see in these terms a reference to baptism and the Lord's Supper. But again, there is no exegetical or contextual reason to associate John's reference to water and blood with those ordinances. It is best to see the "water" here as a reference to Christ's baptism and the "blood" as a reference to His death. Those two notable events bracketed the Lord's earthly ministry, and in both of them the Father testified concerning His Son.
As the forerunner of the Messiah, John the Baptist proclaimed a "baptism of repentance" (Matt. 3:11). He called on the people of Israel to prepare their hearts for the coming of the Messiah by confessing and repenting of their sin, and asking God to cleanse them. John knew that baptism was a public affirmation of repentance from sin and as such, it is an external act symbolizing an internal reality.
However, John the Baptist knew the spotless Lamb of God who takes away the sin of the world had no sin to repent of and hence no need to be baptized. Therefore, John tried to prevent Him from being baptized by saying, I have need to be baptized by You, and do You come to me? (Matt. 3:14). John was shocked and amazed by the reversal of what he knew to be true. He was the sinner, Jesus the sinless one.
Permit it at this time; for in this way it is fitting for us to fulfill all righteousness (Matt. 3:15).
Jesus always performed what God required of His people. His perfect obedience made Him the sinless sacrifice whose death made atonement for sin.
This is My beloved Son, in whom I am well-pleased (Matt. 3:17).
Those words expressed the Father's approval of the Son, and His confirmation of Jesus Christ as the true Messiah.
John then introduces a second witness, the "blood", representing Christ's death. As He had at His baptism, God the Father gave striking testimony to Jesus in the miraculous events surrounding His crucifixion. Matthew 27:45 records that "from the sixth hour darkness fell upon all the land until the ninth hour." (Matt 27:45) In the middle of the day came a supernatural darkness, symbolizing the Father's forsaking of the Son as the sin-bearing sacrifice.
At the moment of Jesus' death there was another astonishing miracle as "the veil of the temple was torn in two from top to bottom" (Mt. 27:51). That curtain, separating the Most Holy Place from the Holy Place, was far too large and heavy for men to tear, especially from the top to the bottom. The Father's act symbolized His acceptance of Jesus' sacrifice and the way into His presence was opened. Our direct access to the Father was established.
Their appearance in bodily form was such an overwhelming and miraculous testimony to Jesus that a Roman centurion who witnessed it cried out, "Truly this was the Son of God!" (Matt. 27:54).
The Father's testimony to Jesus also appears in the Old Testament prophecies that His death fulfilled, most notably in Psalm 22 and Isaiah 53. We will not read those today but, I encourage you to read those chapters this week to see God's testimony of His Son.
Jesus always did the will of the Father in the power of the Spirit.
The witness of the "three that testify: the Spirit and the water and the blood" is in perfect "agreement" and convincingly demonstrates that Jesus is the Christ, the Son of God. How foolish to "receive the testimony of men" about matters of far less significance while rejecting the infinitely "greater … testimony of God … that He has testified concerning His Son." Jesus Christ is the Son of God!
It is the life of the age to come. It is where believers will most fully experience the perfect, unending glory, holiness, and joy of heaven as we relish in perfect unity with God the Father and His Son Jesus Christ.
And there is salvation in no one else, for there is no other name under heaven that has been given among men by which we must be saved.
So the purpose of God's testimony of the Son is an affirmation that all who have been truly believe will live eternally with Him.
"The one who believes in the Son of God has the testimony in himself." Saving faith in Jesus Christ results in a lifelong hold on eternal life. As we have discussed in previous sermons, true faith perseveres. So, those who turn away from the gospel reveal that they were never saved in the first place. They never had a hold on eternal life.
On the other hand, "the one who does not believe God, has made Him a liar". To deny that Jesus Christ is who God said He is, to refuse to believe "in the testimony that God has given concerning His Son", makes God a liar. What an incredulous blasphemy of God!!! We know God is perfect truth and cannot lie. Rejecting God's witness concerning His Son is not something to be pitied, or overlooked in the name of tolerance or political correctness. It is a heinous, damning sin and an affront to God's holy nature.
Here again the exclusivity of the gospel is evident. Only those who believe the Father's witness to the Son and acknowledge Jesus as Lord and Savior have eternal life. All who refuse to do so do not have the Son do not have eternal life.
If you have not accepted Jesus Christ as Lord and Savior, I hope you heard the testimony of God the Father today regarding His beloved Son. He is the Messiah! He is the one and only way to the Father! He is the only way to eternal life in Heaven!


Tells the history of the Nazi's great fortification along the western part of the Channel Coast in what became the D Day invasion area. Describes the massive construction effort involved. Reveals the Allies' ingenious plans and equipment designed to overcome this mighty defensive system.


Everyone loves a mystery. Solve one in science, and accolades are forthcoming. Not so, however, in the realm of the paranormal, where evidence, logic, and theories are often stood on their heads. Whereas forensic scientists, say, begin with the evidence and let it lead to the most likely solution to a mystery, "parascientists" typically begin with the desired answer and work backward to the evidence, employing confirmation bias: They look for that which seems to confirm their prior-held belief and seek to discredit whatever—or whoever—would argue against it.
For example, in the paranormal field of cryptozoology (a term coined by Ivan T. Sanderson to describe the study of "hidden" or unverified animals [Heuvelmans 1968, 508]), proponents of Bigfoot offer a large quantity of evidence. Unfortunately, it is of very poor quality: eyewitness reports, footprint casts, hair samples—just what is attributable to misperception or deception. It is all questionable evidence because, hoaxes aside, neither a live Bigfoot nor a carcass nor even a DNA specimen is available for scientific study.
The same situation holds true for other claims. They include psychic phenomena; ghosts, poltergeists, and demons; flying saucers and aliens; cryptids, such as the Loch Ness monster; spontaneous human combustion; faith healing and weeping statues; the Devil's Triangle; and so on and on. Mainstream science has not verified as genuinely paranormal any of these objects, entities, or occurrences.
Parascientists usually take a different tack. For them, investigation is not a quest to explain a mystery (what they deride as "trying to explain it away") but rather to collect mysteries about whatever paranormalities they believe in, by which they hope to convince others there must be "something to it." In short, they are not detectives but mystery mongers.
For them, the mystery is essentially an end point rather than a beginning. If it is not readily explained, they do not blame a lack of evidence; instead they suppose thereby that something has been established: "We don't know what caused oil to appear on the statue; therefore, it must be a sign from God." But this is a type of logical fallacy known as argumentum ad ignorantiam, an argument from ignorance—that is, drawing a conclusion from a lack of knowledge. One cannot say "We don't know" and then assert that therefore we do know.
And yet that very faulty reasoning is behind most paranormal claims: "We can't explain what caused a; therefore, it's likely b," where a is a hairy-monster sighting or hovering light or an unexpected medical cure, and b is presumed to be, respectively, a Bigfoot, or flying saucer, or miracle. Actually it might instead be, again respectively, an upright-standing bear, Venus seen through layers of atmosphere, or the result of prior medical treatment.
This requirement—this need—for positive rather than negative evidence is ignored or dismissed by the mystery mongers. In the titles of their books and TV documentaries, they trot out such words as unsolved, unexplained, unknown—presenting not mysteries to be investigated and solved but supposedly eternal enigmas that prove (by arguing from ignorance) the existence of the paranormal.
Grant Wilson, who partnered with Jason Hawes in TV's Ghost Hunters, says their approach to ghost hunting was to "end up with only those things you can't explain away" (Hawes and Wilson 2007, 6).
Crop circle advocates have suggested various "theories" to explain the supposedly inexplicable patterns in English grain fields (despite extensive evidence of hoaxing [Nickell 2004, 115–122]). Ken Rogers of the Unexplained Society opines, "The circles are indeed the result of a UFO landing to probe the crops. There is no other explanation . . ." (qtd. in Randles and Fuller 1990, 16).
Perhaps nowhere is negative evidence sought and promoted more avidly than by UFOlogists, whose very subject of study begins with the word unidentified. Prominent among such collectors was Charles Fort (1874–1932). Sometimes considered the "first" UFOlogist (Clark 1998, I: 420), Fort was an armchair purveyor of strange mysteries. Having come into an inheritance that allowed him to indulge his hobby, he spent his last twenty-six years scouring old periodicals for reports of unusual occurrences—including anomalous aerial phenomena—that he taunted "orthodox" scientists to explain (Fort 1941). Not only was his evidence anecdotal and his approach non-investigative, but his "documentation was not always completely accurate" (Gross 2001, 204).
Nevertheless, Fort is the darling of many UFOlogists and other collectors of phenomena that supposedly "defy natural explanation," what they term "Fortean phenomena" or "Forteana," named after him (Guiley 2001, 212–213; Gross 2001, 203–205; Clark 1998, 420–425).
The transformation from skeptic to—no, not believer because that has certain "theological" connotations—a scientist who felt he was on the track of an interesting phenomenon was gradual, but by the late '60's it was complete. Today I would not spend one additional moment on the subject of UFOs if I didn't seriously feel that the UFO phenomenon is real and that efforts to investigate and understand it, and eventually to solve it, could have a profound effect—perhaps even be the springboard to a revolution in man's view of himself and his place in the universe.
Hynek nevertheless grew cautious about the extraterrestrial hypothesis, noting that it "runs up against a very big difficulty, namely, that we are seeing too many UFOs. The earth is only a spot of dust in the Universe. Why should it be honored with so many visits?" Instead, he said, "I am more inclined to think in terms of something metaterrestrial, a sort of parallel reality," positing "that UFOs are related to certain psychic phenomena" (qtd. in Story 2001, 252). Thus he tried to "explain" one unknown by invoking another!
Today, UFOlogists such as Peter B. Davenport, director of the National UFO Reporting Center (NUFORC), believe that the great number of unidentifieds indicate at least something momentous is behind them. Given "impressive quantities of principally eyewitness data," says Davenport, while most eyewitness descriptions are of poor quality, "many of the high-quality sighting reports involve certain objective aspects, which, to an open-minded bystander, are quite impressive." He adds, "Strong evidence suggests that we are dealing with a phenomenon that is being caused by palpable solid objects whose characteristics are not of human design, and whose behavior is suggestive of intelligent control" (qtd. in Story 2001, 150). He is, of course, hinting at extraterrestrials—albeit coyly—while the "objects" remain unidentified.
Hall believes that mistaken observations of terrestrial objects as well as "hoaxes/imagination" are to be rejected as explanations because both are "inapplicable to the hard-core unexplained cases." He prefers instead the possibility of "so-called 'nuts and bolts' visitors from elsewhere" (qtd. in Story 2001, 239).
Investigators of current and historical UFO reports have sifted out cases with sufficient credible evidence to qualify as defensible. These cases suggest that the character of UFO narratives depends in some part on the character of UFO events, and those events owe their character to a source independent of UFO mythology. . . . Even allowing for human fallibility and self-deception, a genuine mystery seems to be left over.
Bullard is clearly relying on the process-of-elimination method that is the basis of negative evidence.
Then there is Stanton T. Friedman, who promotes the notion of extraterrestrial visitation with bluster, smoke screens, and ballyhoo. He rationalizes, "I learned early on that absence of evidence is not the same as evidence for absence." True enough, but then he is still left with absence of evidence. Friedman, a onetime self-described "itinerant nuclear physicist" was fooled by the amateurishly forged "MJ-12 documents" that purported to prove the U.S. government had retrieved a crashed saucer and its humanoid occupants—proof, Friedman believes, that positive evidence has been covered up by a high-level conspiracy (Friedman 1996, 8, 13, 209–219; Nickell with Fischer 1992, 81–105).
The problem with all such grandiose extrapolation from the data is that it lacks positive evidence. No actual flying saucer or extraterrestrial pilot has ever been captured—notwithstanding hoaxes, folktales, and conspiracy claims. There are only eyewitness reports, photos, ground traces, and the like—all of something unidentified.
But don't all those unidentifieds count for something? Well, quantity is not quality. As extensive evidence shows, cases once touted as unexplained were only that; they were not unexplainable, and, as a matter of fact, many of them have since succumbed to investigation. Not one proved to be anything other than a natural or manmade phenomenon—not such classic cases, for example, as Roswell, Rendlesham Forest, Flatwoods, Kecksburg, Exeter, Phoenix, and Stephenville (Nickell and McGaha 2012; McGaha and Nickell 2011, 2015). Some cases may never be explained because of original eyewitness error, falsified evidence, lack of essential information, or other flaws. For similar reasons some murders remain unsolved, yet we do not consider those cases evidence of a homicide gremlin.
Nothing said so far means we should not continue to investigate unexplained phenomena, including UFOs. After all, onetime skepticism of fiery stones falling from the skies ultimately gave way to proof of meteorites. Science has nothing to fear from the examination of UFO reports, which, to date, have not been useless after all: We have learned much about illusions and misperceptions and fantasy, about personality traits, about rare phenomena such as ball lightning, about the propensity of immature persons to perpetrate hoaxes (skeptics included!), and much more. But investigation must go beyond just collecting negative evidence. It must represent a real attempt to solve—that is, to explain—a mystery.
Clark, Jerome. 1998. The UFO Encyclopedia, 2nd ed. (in two volumes). Detroit, MI: Omnigraphics.
Fort, Charles. 1941. The Complete Books of Charles Fort. Reprinted New York: Dover, 1974.
Heuvelmans, Bernard. 1968. In the Wake of the Sea Serpents; trans. Richard Garnett. New York: Hill and Wang.
Hynek, J. Allen. 1977. The UFO Report. Reprinted New York: Barnes & Noble, 1997.


I am convinced that cyber bullying is worse compared to traditional face-to-face bullying. The threat posed by cyber bullying is a lot more destructive for children being attacked. It results in many complications of victim's life. Cyber bullying has come to displace the traditional face-to-face bully. It poses a threat of social implications (Mair, 98). New technology has enabled cruel individuals to hide behind it. Cyber bullies develop fake identities that allow them to hide, but still attack innocent users on the Internet. It is now more dangerous as compared to facing the attacker. This secret way of communication is able to ensure anonymity of fighting individuals. The intention of the threat is felt, but the addressee cannot determine the source of the threat.
Cyber bullying in this case can cause harm that cannot be felt during the traditional face-to-face bullying. When a victim of face-to-face bullying is attacked, the source of threat can be determined clearly disclosing the authenticity of the attack (Carlson, 34). The old-fashioned form of threatening could not enable many people to share the same network zone to conduct their attacks. The strive of parents to ensure the presence of censorship is caused by cyber bullying. Parents and even guardians continuously monitor the content made accessible by such technology that might compromise safety of their children. The use of technological gadgets (like cell phones and iPods) is discouraged. Parents even go a step further when they provide their children with mobile phones that closely monitor their activity. This is a significant step towards outdoing the wits of the bullies who bother other people. Face-to-face bullying can only be felt during the direct contact with the bully. Conversely, it cannot be felt when the contact is indirect. In such situations, dealing with a bully is much easier. On the other hand, meeting, trapping, or catching a cyber bully is difficult. It is clear that there is fear instilled in the hearts of parents and guardians. Both face-to-face bullying and cyber bullying instill fear. Measures to advance surveillance of parents are a result of fear for the safety of their children.
Cyber bullying has psychological implications that arise from long term effects on the victim. Unlike the direct contact with a bully, cyber attack is able to haunt the victim for a prolonged time, especially when it takes place online. Traumatic feelings are felt by the victim because of the harassment directed at him/her. These might include psychological complications like depression, anxiety, and stress pile up, after which the victim might suffer psychological breakdown. Compared to old-fashioned bullying, which could only affect physical and emotional state of a victim for only a short time in child's life span, cyber bullying can even prompt suicide. Students who have had an experience with this type of bullying have shown reduced and poor interest in social life as well as emotional stress, effects of which are fatal. Based on this, cyber bullying is worse than face-to-face in its context. Depression is more often recorded among victims of cyber bullying, which is quite opposite to the traditional way, where both the victim and proprietor suffer (Rosenthal, 1998).
Cyber bullying is easier to perform for the proprietor. It can be carried out at any time from whichever location and still maintain anonymity of a bully. It is entirely opposite to traditional bullying, which occurs at specific location and time. The bully carries out the attack in a defined area and the timing of act must be suitable for effectiveness. Playgrounds and street corners are the locations where this traditional bullying takes place most often. Though cyber bullying allows a no escape for the victim. No aspect of evasive plotting is practical because the victim can be subjected to the perpetrator's manipulation any time.
Traditional bullying or face-to-face bullying is deadly when compared to this contemporary form of bullying in terms of credibility. It becomes extremely easy for a perpetrator to follow the victim, get the address, and later attack that shows where one resides a serious approach will be undertaken. An example is an attack on the family where it prompts the victim to react quickly, such happens in extreme cases. Consider a direct threat of the attack on a victim at an unspecified time and place. It will certainly eat the conscience of the affected person. They will have to report this to the authority or even seek refuge elsewhere. These actions prove quite effective because the proprietor is assumed to easily reach the victim and fulfill his intentions.
Academic work suffers a powerful punch when bully kids spend a lot of time on the Internet in the quest to threaten their victims. Studying is given little time, and this results in poor performance at school and decreases interest in education. Despite being present at school, bullies still participate and indulge in this activity. This behavior moves from being fun and turns into addiction.
Looking at the aspect of affordability, it is critical that not every individual can access necessary gadgets to perform bullying activities. Due to different economic status, not everyone can afford these modern luxuries. During face-to-face bullying the proprietor can easily carry out their attacks because they need minimal facilities as compared to the use of computer and other communication system. Due to this technological growth, these bullies can launch their attacks quickly, unlike those who use face-to-face confrontation that needs planning (Bryant-Mole, 2009). Cyber bullying is, therefore, not as time consuming as compared to the traditional approach.
Passing of e-mails or pictures bearing threats by bystanders online makes them bullies as well, although their actions might be unintentional. Another key issue is that attacks of the 'weak' kids can persist for a long time. This is different from the traditional way that does not last long. Bullies, who use the traditional method do not spend a lot of their time in public posing to be dangerous to others. Face-to-face bullying does not support other party's involvement, making the activity personal.
Sending of messages or texts with materials that are perceived as attacks on others leave a trace behind. It becomes easy for information regarding threats or malicious attacks to be located and used against the sender (Sanders, 1996). This is a disadvantage of cyber bullying when compared to the traditional face-to-face bullying, which does not leave traces behind.
The only proper way to curb this problem is for parents to take responsibility for activities of their children. This is because majority of gadgets used in cyber bullying are bought by them. It will reduce cases of modern cyber bullying.


text:
By INDRAJIT BASU in Kolkata
China Daily Asia Weekly February 18-24, 2011
Unlike many countries in the world, India has fought off the scourge of the global economic crisis that has resulted in large-scale unemployment. The rate of decline in employment is minor. But there lies a vast gray area in a country where countless young people are still struggling to be gainfully employed.
India is saddled with this problem even as its scorching economy has started spewing out thousands of new opportunities, and a growing number of young people are willing to grab anything on the table.
According to experts, unemployment is almost like an unexploded bomb, and despite many eff orts, the country still has some way to go. A recent research by Business Standard, a publication group, reveals that while India's business sector has added close to $300 billion of fixed assets over the past decade, labor costs have remained flat at about 6.5 percent during the same period. "Since wages and salaries have increased across the board, it implies that recruitment in manufacturing fi rms is either fl at or may even have declined slightly," the study says.
Similarly, a survey by the country's labor ministry for the period October- December 2009 said although the overall scenario improved by about 628,000 jobs during December 2009 over September 2009, in the estimated total labor force of over 520 million in 2009-2010, as many as 9.4 percent remained unemployed nationwide, and it was 10.1 percent in villages where 60 percent of the country's people live.
"Such numbers, however, provide a partial picture, as they cover only selected industries and relate mainly to formal employment," says a recent International Labour Organisation report.
"About 86 percent of India's workers are in informal employment and over 90 percent are in what is known as the unorganised sector. These shares have changed little in recent years, indicating that India's strong and steady growth has not yet yielded a signifi cant improvement in terms of broadening access to quality employment." A bleak example of the crisis is a recent incident in a small northern Indian town where more than 100,000 people gathered to apply for 416 jobs offering a mere Rs 5,200 ($115) a month.
The oddity is not so much for its massive turnout for so few vacancies in "lowly positions" such as dhobi or washermen, barbers and water carriers, but for the violence that erupted subsequently when applicants grew frustrated.
"While India has seen resumption of rapid economic growth post crisis, employing youth (aged between 19 and 35 years) is still a big problem," says Rohit Krishnan, an industry expert at London Business School. "Its eff ects are visible at every segment of society – from the economics of labor market to social unrest, to political dissatisfaction and crime."
"I think when we are looking at the growth scenarios being outlined for India, we are talking about 8 to 9 percent growth over the next 10 years. And then when we look at the demographic projections, we see that we are going to have a very young labor force," says International Labor Organisation's Director and Country Offi cer, India, Tine Staermose. According to her, the average age of an Indian by 2020 is estimated to be 29 years compared with 37 years in China and the United States, 45 years in Western Europe, and 48 years in Japan. "Undoubtedly that is a huge challenge, which brings the urgency to ensure that this labor force is properly skilled to be able to fi nd jobs, and to be provided with decent employment," she says.
"India's unemployment problems stem primarily from the unprecedented phase of demographic changes," says India's Ministry of Labour and Employment Secretary Prabhat C. Chaturvedi. These changes, he adds, contribute to an ever-increasing size of labor force. According to projections, the proportion of population in the working-age group (15-59 years) is likely to increase from approximately 58 percent in 2001 to more than 64 percent by 2021.
Youth unemployment in urban areas arises from the shift of a large number of rural youth, in search of jobs, to urban areas, and (also the fact that they) don't find it because of lack of skills that the industry seeks, and lack of the industry's ability to utilize the skills they have," says Rohit Krishnan.
According to Mukesh Gupta, a specialist at ILO's South Asia offi ce, "The overcrowded agricultural sector is also a big contributor to employment woes." With the shift from agriculture to other vocations being very low, a large number of people are either unemployed or underemployed. Chaturvedi says India has initiated a number of programs for skill development over the past few years. "Our target is to have 500 million trained youth by 2022. Most notable schemes for creating wage employment are the National Rural Employment Guarantee Programme (NREGP) in rural areas and Swarna Jayanti Shahari Rozgar Yojana (SJSRY) in urban areas," he adds. NREGP, which was launched in 2005, aims to provide every household with at least 100 days of guaranteed wage employment in a year. "This is India's most far-reaching eff ort to guarantee wage employment. It provided gainful employment to 45 million people in rural India last year," says Gupta. SJSRY on the other hand seeks to provide jobs to the unemployed and underemployed poor in cities. It also helps them set up self-employment ventures. This scheme, says Chaturvedi, was revamped last year with an idea to encourage the private sector to invest in skill development eff orts. ILO adds that the government and private sector's pursuit to improve skills development and training could result in the number of people covered under skill development programs rising from 3.1 million to more than 10 million a year in the next few years. This may provide light at the end of the tunnel for many, even though the tunnel may appear long. "The good part is that India will continue to grow between 7 and 9 percent for the next two decades. With all the eff orts that both the government and the private sector is taking, India is going to look totally diff erent from what it has been in the last 200 years," says Nirupama VG, a human resource and recruitment consultant, and the founder and managing director of Ad Astra Consultants. "India has hardly experienced any structural growth in the past. But all that is changing. While the private sector has been very dynamic [in the past decade], the government too is catching up. And together that can make a big difference," she adds
C level hiring is on in full swing

redpajama_set_name: RedPajamaCommonCrawl


text:
ABT Studio Company/Royal Ballet School: Emerging Offerings
ABT Studio Company and Graduates of the Royal Ballet School
NYU Skirball Center
Joint Program:
Birthday Offering (excerpts), Kabalevsky Violin Concerto, Concerto Grosso, Chromatic Fantasy, Concerto (pas de deux), See the Youth Advance!, New Scarlett
In 2003, the New York-based American Ballet Theatre Studio Company and London's Royal Ballet School began an exchange program during which dancers periodically visit each other's schools, live in each other's homes, take master classes and coaching sessions from the other group's teachers, and generally feed off their mutual artistic development. This exchange program continues, and the fact that it has lasted as long as it has speaks to its value for the participating young dancers.
On various occasions during exchange visits, each group presents a program to display their young dancers' progress – as well as to entertain audiences who come to see the cream of the crop of future dancers with ABT, The Royal Ballet, and other professional companies. On Friday and Saturday at NYU's Skirball Center, the dancers of ABT Studio Company and recent graduates from the Royal Ballet School presented one of their joint performances. The program included six dances that separately featured each group's future professionals (three each), and one, a new piece choreographed by Liam Scarlett, that combined dancers from the two groups. I saw Saturday night's program. Although the choreography and execution was somewhat uneven, and some of the choreographic choices questionable, it proved to be a fun way to get introduced (or in one case, reintroduced) to these young dancers.
However, one overriding issue clouded the program, at least for me. For any "first contact" with dancers, it's difficult to identify them unless they're named specifically in featured roles, or at least listed as participating in a given piece. RBS elected not to identify its dancers in any dance, except for those in the combined Scarlett piece, and ABT Studio Company inexplicably omitted dancer identification in one of the pieces they performed. Consequently, what I (or anyone in the audience who doesn't already know them) can gather of a dancer's identity must come from a process of elimination or online research, and is uncertain. It makes praise or critical commentary difficult at best. I understand that casting flexibility may be necessary in pieces involving large numbers of dancers, but surely there was sufficient time to have provided program inserts.
I'll discuss the evening's highlights first.
For me, the evening's strongest piece was Marcelo Gomes's Kabalevsky Violin Concerto, which had its New York City premiere the previous night. It's a delightful piece of work for two couples that plays on the details in Dmitri Kabalevsky's Concerto for Violin in C Major, Op. 48 without losing sight of the unity in the composition.
Zimmi Coker and Luigi Crispino
of ABT Studio Company
in Marcelo Gomes's
"Kabalevsky Violin Concerto"
Photo by Rosalie O'Connor
Kabalevsky Violin Concerto could have been merely an exercise in translating the composer's simple-sounding music into simple-looking visual images, but it's more than that. Gomes's choreography, though not complex, sufficiently challenges the dancers and provides variety for the audience. And his choreography here is what I describe as circular rather than linear – it doesn't simply end (which might be appropriate in certain circumstances); it brings the different segments together, and does so in a surprising way that tickles the mind and provides an unexpected visual punch. It's especially rewarding to see how Gomes has progressed as a choreographer over the years; he's developed not only a fine choreographic sense, but, equally important, sensitivity both to what works best for a piece of music and for the dancers in it, and what will be satisfying to an audience.
Like the composition, the ballet is divided into three parts: the first is filled with playful, quicksilver movement that mirrors Kabalevsky's playful, rapid-fire rhythms; the second movement is lyrical and fluid and as enchanting as a fairy tale pas de deux; and the third combines aspects of the first two.
Lea Fleytoux and João Menegussi
At this performance, the first two movements were danced respectively by Lea Fleytoux and João Menegussi, and Zimmi Coker and Luigi Crispino, and in the third they dance together (sometimes switching partners; sometimes not). Fleytoux and Menegussi danced with precision and a sense of mercurial friskiness, without sacrificing form. If the opening piece on the program (which I'll get to later) tended to leave an audience, or at least me, feeling benumbed, these two dancers provided a shot of visual adrenaline.
In the second movement, Coker and Crispino took the piece to an even higher level.
Photo by Gillian Murphy
Even if I hadn't already seen and remembered her from a prior ABT Studio Company performance, Coker would have been instantly noticeable, and hard to ignore. With her shock of red hair and a vivacious stage personality to match, she's a pleasure to watch. She's the type of dancer who already can light up the stage and at the same time melt the heart, and she has an endearing and somewhat mischievous quality (befitting her first name) that people would pay to see – and I suspect that ABT knows it. There's still stuff to work on – to nitpicky me, she looked somewhat round-shouldered, indicating the need for upper body work – but the equipment and talent is all there. And Crispino, who I'd not previously seen, is already a gallant, commanding, and highly capable partner, with a built-in bit of heartthrob. And it's a measure of the strength and versatility of these dancers that at Friday's performance, Coker and Crispino danced the first movement.
Not to be outdone, RBS included a duet in the program – the pas de deux from Sir Kenneth MacMillan's Concerto – that highlighted (at times literally) two of their finest. Even as an excerpt, it's a wonderful example of MacMillan choreography. It has all the rapture of his Romeo and Juliet pas de deux, but without the feverish passion (the full moon against which the dancers' shadows were sometimes projected – designs by Jürgen Rose – provided all the atmospheric passion necessary). And as executed by Sae Maeda and Joshua Jumper, the piece found very fine interpreters. Maeda has an extraordinary pristine line and extension, and her air of serenity fit the piece exquisitely. Although there were one or two noticeable glitches, Jumper overall did excellent work partnering her while making his portrayal both ardent and credible.
Sae Maeda and Joshua Junker,
graduates of the Royal Ballet School,
in the pas de deux from
Sir Kenneth MacMillan's "Concerto"
Scarlett's new ballet closed the program, had its world premiere the previous night, and looks a bit like a work in progress – which might be reflected in the fact that the piece has no name: it's called simply "New Scarlett." But even if it is a work in progress, it already has lots to recommend it.
Choreographed to the Third Movement of Philip Glass's Piano Concerto No. 2, "New Scarlett" looks remarkably polished for a piece that was stitched together on separate sides of the pond, with the dancers being given only a week to work on it together. Indeed, while it lacks the complexity of his dance for New York City Ballet (Acheron) and the senselessly arrogant audacity of his piece for ABT (With a Chance of Rain), it has the authority and energy of the ballet he created for Miami City Ballet (Viscera), which remains his finest work of those I've seen.
Sae Maeda and Albjon Gjorllaku,
in Liam Scarlett's "New Scarlett"
At first, "New Scarlett" appears to have a structure of little structure; of being strange for the sake of being strange. It's a large piece, consisting of six men and five women, and initially looked more gimmicky than anything else (e.g., with one dancer facing backwards while others dance for no apparent reason). But soon these images begin to look meaningful and structurally, as well as visually, significant. The dancers shift seamlessly between seemingly unrelated subgroups (mostly pairs and solos) as the piece evolves. That's not particularly unusual. But the way that Scarlett cleverly weaves his dancers into and out of various groupings, and his facility with presenting different groups dancing concurrently on different parts of the stage, but to quite different choreography (not just in counterpoint or movement syncopation), yields a glorious visual tapestry. There may have been an arithmetic or geometric progression going on, but one viewing didn't allow for certainty – and whether it did or not isn't really significant. That it maintains visual interest is.
My only concern was the unevenness of the division between the men and women– which resulted in a somewhat awkward looking imbalance and sense of 'odd-man-out-for-no-reason' when all appeared together at the dance's conclusion (although perhaps that relates back to the 'one dancer facing backward' at the piece's beginning). The listed dancers (Sierra Armstrong, Virginia Lensi, Coker, Mattia Santini, Crispino, and Menegussi from ABT Studio Company; and Alice Bellino, Maeda, Sean Flanagan, Albjon Gjorllaku, and Augustus Payne from RBS), all did excellent work.
Graduates of the Royal Ballet School
in Helgi Thomasson's "Concerto Grosso"
Concerto Grosso, choreographed by Helgi Tomasson to music by Francesco Geminiani (La Follia [after Corelli]) is a powerful dance for five men, here performed by the RBS graduates. Overall, it's a liberating type of piece – highly athletic but not without form or substance, and it gave each young man a chance to shine. One, wearing a red costume, was the piece's central figure. Except for overshooting his turn at the end, he did a fine job – and made up for deficiencies in his partnering that I noticed in the opening piece. [It may not be fair comment at this point, but partnering generally was not as strong a quality with the RBS's young men as it was for ABT's.]
The other pieces on the program were more problematic.
Ethan Stiefel's See the Youth Advance! is perfectly serviceable, but not particularly memorable. It was commissioned as a joint work for the dancers from the two schools (the first jointly performed piece in the exchange program's history), premiered in London last year, and had its New York premiere during this engagement, danced just by unidentified members of ABT Studio Company.
Sierra Armstrong and Jarod Curley
in Ethan Stiefel's
"See the Youth Advance!"
What significance there is to the piece is flagged by its title, and by the music to which it's choreographed. In 1746, Handel composed an oratorio in three acts titled Judas Maccabaeus. That piece's third movement is subtitled See the Conqu'ring Hero Comes!, which subsequently achieved a popularity of its own separate and apart from that of the oratorio. Later, Beethoven became one of Handel's pupils. In 1796, in the process of proving himself to the musical world (and wealthy potential benefactors), the 26 year old Beethoven composed 12 Variations on his idol's 'See the Conqu'ring Hero Comes!' So the music represents young Beethoven's effort to show how much he'd learned, and how great a talent he was. It seems logical to me that Stiefel chose this music as an apt vehicle to make the same statement about these dancers.
Zimmi Coker and João Menegussi
Stiefel's choreography isn't as significant as the music in that respect, but it gets the job done. After an opening minuet-like dance (more formal than folk; rougher than a court dance – perhaps representative of the students' raw talent), the piece evolves into variations of varying length and substance involving subgroups – mostly pairs. I didn't see any choreographic evolution (a la Jerome Robbins's The Goldberg Variations), but that would have been beyond this piece's scope. Aside from those segments in which Coker was involved, the piece's highlight was the work by Armstrong and Jarod Curley (who bears a remote resemblance to David Hallberg, and shows considerable promise).
Choreographed to the Chaconne from Chromatic Fantasy by Dave Brubeck, Dana Genshaft's Chromatic Fantasy, which also had its New York premiere at Friday's performance, doesn't seem to know exactly where it's going or if it's going anywhere. Performed by six dancers from ABT Studio Company, it looks like Genshaft was trying to reflect the jazziness in Brubeck's score (no surprise there), but the choreographic palette is limited, and the imagination even more so. And opening with the dancers upstage center in a scrum illuminated by an overhead light, then scattering in the darkness and replaced by individual dancers going through the motions, and ending not with a return to the scrum, but in a completely different and unsatisfying way, left a sour impression. The six dancers (Kiely Groenewegen, Armstrong, Lensi, Andrii Ishchuk, Javier Rivet, and Santini executed well enough, but couldn't overcome the dance's dullness – for which the color-block costumes by Reid Bartelme and Harriet Jung overcompensated.
ABT Studio Company dancers
in Dana Genshaft's "Chromatic Fantasy"
Which brings me back to the beginning. The evening opened with an excerpt from Sir Frederick Ashton's Birthday Offering. I've frequently written that I have a prejudice against excerpts unless they can stand on their own (like the pas de deux from Concerto), and this one can't. And even though it's elegant fluff (it was created in 1956 for the celebration of the Royal Ballet's 25th Anniversary), it looks its age at least based on this excerpt: as dated as a birthday cake that's been kept in the freezer too long. The piece as a whole was reportedly intended as somewhat of an homage to Petipa, but, again based on this performance and this excerpt, it's more form than soul. Compare similar homages to Petipa and the Russian Imperial Ballet created by George Balanchine. Including an Ashton piece on the program was not inappropriate – his body of work is one of the pinnacles of the ballet in general and, of course, The Royal Ballet in particular, but a different piece might have looked less starched.
Be that as it may, the unidentified RBS dancers did well with it, but with a few exceptions (the lead couple; and one dancer, I think Amelia Palmiero, who seemed more vivacious than the others but was consistently being pulled off – or not maintained on – center) the execution generally looked more reverential than robust. The best part were the ornate "birthday cake" costumes by Andre Levasseur. The program improved considerably thereafter.
Tags: ABT Studio Company, The Royal Ballet School
Emerging Choreographers Program: Inner Landscape
New York City Ballet: The Sleeping Beauty
The Royal Ballet Online Part 1 Updated
Cherylyn Lavagnino Dance: Mythbusters
The Royal Ballet School: Summer Performance 2014
Botis Seva: BLKDOG
Ballet BC: The Cutting Edge
Twyla Now, And Then


text:
Your Guide to Writing a Convincing Villain
Character, Writing Advice
My first foray into villainy was Saturday morning cartoons, specifically those darn Wile E. Coyote and the Roadrunner cartoons. That crazy coyote never gave up and was obviously pegged as the villain.
Although I got the sneaky suspicion that the audience was supposed to hate the coyote, I just couldn't. He was surrounded by a palpable desperation and motivated by an unrelenting pride-- both of which I could understand and relate to on a visceral level. Sure, he was the villain, but he was also relatable.
He was a coyote, a killer in ruthless pursuit, a villain. As a carnivorous animal, it was in his nature to be in direct conflict with his prey. But from Wile E. Coyote's perspective, he was hungry and this roadrunner was evidently the only morsel of food in the desert. Too bad Acme didn't sell food, too.
Image Courtesy of Giphy
To create a convincing villain, your villain must have the following:
Motivations that the reader can understand
Moments of relatability that make the villain vulnerable and tragic
Creating an all evil, all of the time character will fall flat. The most fearsome, formidable foes are round characters living out their own lives. They may do horrible things, but they also have tender moments (even if it's tucked into their backstory).
So, let's talk about how to create a multi-dimensional villain that's worthy of your story.
Here's your helpful list of best practices for writing the perfect villain.
BEST PRACTICES FOR WRITING VILLAINS
Here's a list of best practices for writing villains. Subscribe to receive this extra resource.
What is a Villain?
You need a protagonist to tell your story and move it forward. But you need a villain to thwart and develop your protagonist.
You may be tempted to define the villain as "the bad guy", but that description is a little too shallow and one dimensional if you really want to create a compelling character.
A villain is someone who comes into direct conflict with your protagonist. The two are on a collision course.
What makes the villain "bad" is that he's opposing your protagonist.
But it's also important to note that a villain does not view himself as a villain. It's only the protagonist of your story (and, by extension, the reader) who perceives and labels the villain as such. From the villain's perspective, he is the protagonist of his own story. It's essential that you, as the writer, don't forget that.
From the villain's perspective, he is the protagonist of his own story. Don't forget that.
Writing the villain with the consciousness that he is actively involved as the hero of his own story will make your villain more realistic, interesting, and compelling. The villain stops being the obligatory bad guy, and becomes the opposing force on a collision course with your protagonist.
Think of it this way: you have two stories headed by two protagonists that just so happen to intertwine. You as the writer choose to follow one of protagonists. From the eyes of your chosen protagonist, the other protagonist becomes villain.
Different Types of Villains
Depending on who you ask, there are countless different types of villains. For the sake of simplicity, let's divide villains into two types: human and abstract.
A villain can be human (or have human qualities). He or she is a person (or a collection of persons) that prevents your protagonist from achieving his goal. The term "human" can extend to anthropomorphized animals, aliens, etc.
A villain can be abstract. The villain is not embodied in physical form. The villain can be a setting, a society, or even the protagonist's self. Your story's antagonist can be a force.
Be careful with abstract villains. They don't elicit the same "visceral" response in a reader as a personified villain. If you choose to go with an antagonistic force, assign a person to represent the abstract so that it gives your protagonist (and your reader) something tangible to loathe.
The Best Type of Villain Is…
You've heard it before: create a person that the readers will love to hate, but I think the genius villains are those the readers hate to love-- the ones you feel bad for, and then you feel bad for feeling bad.
To me, that marks a well-written villain.
In my opinion, the best type of villain also serves as a foil. He or she is opposite in action, demeanor, motivation, and delivery to the protagonist. By thoughtfully characterizing the villain, you're also providing a sharp contrast for the protagonist. The reader can understand certain truths about the protagonist that's only brought out by comparison to the antagonist.
The hero is usually pushed into the role of hero by the villain. The protagonist cannot be a hero without the villain.
Also note that the villain may also negatively mirror the protagonist. The two may share some of the same characteristics, or have a similar backstory.
All Realistic Villains Share This in Common
As we discussed before, villains are the hero of their own story. They're doing what's right to them based on their own (twisted?) moral code.
That's why it's crucial to craft a comprehensive moral code for your villain. The point of this exercise isn't to create a moral code that matches your own, but it's to create a framework that your villain will operate within. Even if you don't explain the villain's moral code to your reader, it's important that you know it and your villain never violates it.
List what your villain stands for, and what he or she will do and will never do.
A good example of giving the villain a moral code was written into Cormac McCarthy's No Country for Old Men. Hitman Anton Chigurh is one of the most chilling villains of all time, but he was slave to his own moral code: whether a person lived or died was determined by the flimsiness of fate (in a coin toss). It doesn't get scarier than that.
Allow the Villain to Win
The reader needs to know that the villain means (and has the means to do) exactly what he or she threatens to do. Prove that the villain is capable of bringing pain to the protagonist. The easiest way to do this by showing the villain victimizing others of similar characteristics to your protagonist.
Otherwise, such threats ring hollow.
As your story progresses, you need to develop increasing tension between the villain and the protagonist. The reader must be in constant fear that the villain will win, and you'll do that by giving the villain small wins along the way. Here's more on what tension is and how to develop it in your story.
Crafting a Backstory for Your Villain
The best villains are born out of a hopelessly tragic, soul crushing backstory. If you can figure out how to craft a villain's backstory that moves the reader and makes them feel like, Wow, I actually get it-- the villain is making horrific choices, but I still get why he feels this way -- you've done your job.
You need to create sympathy for the villain, too. Even if it's a moment of sympathy that's quickly snatched away.
If you can't find sympathy for your villain, rethink your villain because you're in danger of creating a one-dimensional character.
Finding sympathy for the character doesn't mean that you must agree or approve of who the villain is now.
Also, give your villain a motivation. His sole motivation shouldn't be to thwart the protagonist. That may be a "fun" bonus for the villain, but his motivation is likely to win or validate himself or complete a mission.
Create an Arc for the Villain
Develop a story arc for your villain, too. Your antagonist should be involved within his own his own story-- a story that the protagonist interrupts.
Don't just insert the villain whenever your protagonist needs it. Continue to document his story, even if you don't include it in your narrative, so that the timeline, motivations, and pacing makes sense. This is how you create depth to your villain.
Avoid Creating Caricatures
Even with the best intentions, you could create a caricature, and not a rounded character. It's easy to get carried away. Your goal may be to illustrate the villain's dark side; however if you're not careful, the villain can read as disproportionately evil.
Your villain should not be all bad all the time. In fact, he or she should have some good qualities. Perhaps he's funny, charming, and inspiring (to his minions). Perhaps she's exciting, talented, and clever.
A well-written villain is not simply an exaggeration of evil qualities personified.
Watch Your Language
Don't make the villain sound too villainy. Avoid the temptation to give your villain flowery language that doesn't sound like the rest of the characters in your story.
Your villain doesn't need to sound eloquent at all. He or she should sound like a product of his environment and world.
If your villain sounds like he was lifted off the pages of a Sherlock Holmes novel, but you're writing a novel set in the 22nd century, something's off. It reads inauthentic and reduces the villain to caricature.
To create a villain your readers will care about, you'll need to define how he is the hero of his own story. If you reduce the villain to a bad guy without taking time to understand from his perspective, you'll cheat your reader.
The best characters, villains included, are complicated and messy.
Don't forget to download this list of best practices for writing villains.
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Modified proteins were detected in liver and bone marrow of mice following treatment with [14C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable 14C/13C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of 14C. A number of proteins were identified by in-gel proteolysis and conventional mass spectrometric methods with the low molecular weight proteins identified including hemoglobin and several histones. The incorporation of 14C was largely proportional to the density of gel staining, giving little evidence that these proteins were specific targets for selective labeling. This was also true for individual histones subfractionated with Triton-acid-urea gels. A representative histone, H4, was isolated and digested with endopeptidase Asp-N, and the resulting peptides were separated by high performance liquid chromatography. 14C levels in collected fractions were determined, and the peptides were identified by conventional mass spectrometry. The modifications were distributed throughout the protein, and no particular amino acids or groups of amino acids were identified as selective targets. Thus chemical attack by one or more benzene metabolites upon histones was identified and confirmed, but the resulting modifications appeared to be largely nonspecific. This implies high reactivity toward proteins, enabling such attack to occur at multiple sites within multiple targets. It is not known to what extent, if any, the modification of the core histones may contribute to the carcinogenicity of benzene.
Human exposure to benzene occurs both occupationally in the chemical and fuel industry and environmentally through automobile exhaust, gasoline, and cigarette smoke (1). Chronic exposure to benzene is known to cause aplastic anemia and an increased risk of acute myelogenous leukemia in humans (2, 3). High exposures result in toxic responses in rodent bone marrow, including aplastic anemia, micronuclei, leukocytopenia, and hyperplasia (4–6). Long term animal studies show that benzene causes tumors at multiple sites in mice and rats (4). However, the effects of the numerous electrophilic metabolites of benzene and molecular processes underlying hematotoxicity and leukemogenesis remain unclear.
Benzene is oxidized in the liver by cytochrome P4502E1 to form benzene oxide (7–9), which gives rise to phenol, catechol, hydroquinone, 1,2,4-benzenetriol, and ring-opened metabolites such as trans-trans-muconic acid (10, 11). It is the reactive metabolites of benzene that are responsible for myelotoxicity. Hydroquinone and catechol concentrate and persist in the bone marrow after benzene exposure (12, 13), and significant levels of muconic acid and glucuronide and sulfate conjugates of hydroquinone and catechol are also found (14). Hematotoxicity and leukemogenicity occur upon transport of hepatic phenolic metabolites to the various bone marrow cell populations where they are oxidized further via a peroxidase-mediated reaction to their quinone and semiquinone derivatives (15–18). Various combinations of benzene metabolites can exert a synergistic effect on multiple cellular targets leading to increased toxicity (19–21). Bone marrow is a complex tissue containing hematopoietic stem cells and stromal cells, both of which are potential targets of benzene and its metabolites. There are likely multiple cellular and molecular targets of the metabolites in the bone marrow compartment that are involved in hematotoxicity and carcinogenicity (22). Developments in the assessment of risks associated with exposure to benzene have been reviewed recently (23, 24).
Benzene is known to cause aneuploidy but is not a strong DNA binding agent and is only weakly mutagenic (25, 26). We previously determined that [14C]benzene, administered at extremely low doses in mice, bound to proteins at a higher ratio compared with DNA in the target organ, bone marrow (25, 27). Elevated levels of benzene oxide and hydroquinone adducts of hemoglobin and albumin have been observed in workers subject to benzene exposure (28, 29). It could be valuable to identify any other protein targets of benzene metabolites in the bone marrow to further our understanding of benzene-induced leukemogenesis.
Accelerator mass spectrometry (AMS)1 provides an extremely sensitive method for the detection of low level modifications of proteins (30). We have utilized this technique to develop a powerful strategy for identification of target proteins of isotopically labeled xenobiotics in vivo, combining AMS with high resolution two-dimensional gel electrophoresis, Triton-acid-urea gels, and HPLC to separate complex protein mixtures and biological mass spectrometry (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and LC-ESI-MS) to identify polypeptides bearing 14C modifications. In addition to confirming previous reports of hemoglobin as a target for modification by benzene (30, 31), we also report the identification of the core histones as targets for [14C]benzene metabolites detected at attomole levels in microgram amounts of mouse bone marrow protein.
[U-14C]Benzene (specific activity, 58.2 mCi/mmol, corresponding to approximately one atom of 14C per benzene molecule) was obtained from Sigma. Radiopurity was found to be >99% by HPLC. The dosing solution was prepared in filter-sterilized corn oil by dilution of the stock [14C]benzene. The dosing solution concentration was verified by liquid scintillation counting. All chemicals were analytical or electrophoresis grade and obtained from Amersham Biosciences or Sigma.
These investigations were conducted under established federal regulations for the care and use of laboratory animals and were approved by the Lawrence Livermore National Laboratory Animal Care Committee. Male B6C3F1 mice, each weighing 22–25 g, were purchased from Charles River Laboratory and allowed to acclimate in an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility for a minimum of 1 week before use. Animals were housed three to a cage in filter top, polycarbonate cages with hardwood chip bedding. They were maintained on a 12-h light/dark cycle at ≈22 °C and received laboratory chow and water ad libitum. Each animal received a single dose of corn oil (controls) or corn oil containing [14C]benzene via intraperitoneal injection. Each dosing stock was verified by liquid scintillation counting prior to administration. Two dosage regimens were used: 155 μg/kg of body weight, sacrificed at 1.5 h post-treatment, hereafter referred to as "low dose"; and 800 μg/kg of body weight, sacrificed at 18 h post-treatment, referred to as "high dose." All mice were euthanized by CO2 asphyxiation. The 18-h time point was selected based on previous time course data (25, 27) showing maximum adduct levels in the bone marrow at this time point.
The humerus and femur bones were dissected from each mouse using disposable scalpels and forceps (those from the control mice being collected first) to ensure no 14C cross-contamination occurred. Tissues were not perfused to remove residual blood. The marrow was flushed from the bone shaft with 2 ml of phosphate-buffered saline, pH 7.4 using a disposable 26-gauge needle. Bone marrow from each treatment group was pooled, and the cells were washed three times in phosphate-buffered saline and centrifuged at 1000 × g. Cells were lysed in 9 m urea, 4% CHAPS, 65 mm dithiothreitol, 2% ampholytes (Pharmalyte 3–10), 10 mm spermine, 40 mm Tris base and then centrifuged at 450,000 × g for 10 min at 22 °C in a Beckman TL100 tabletop ultracentrifuge to remove DNA. Protein concentrations in the supernatants were determined by a modified Bradford assay (31) using the Bio-Rad protein assay kit and ovalbumin as a standard. Samples were stored at −80 °C prior to electrophoresis. Livers were dissected, cut into small pieces, and washed twice in 20 mm Tris-HCl, pH 7.4. The tissue was homogenized in 3 volumes of the same buffer and centrifuged at 9000 × g for 20 min. The supernatant was lysed with 2 volumes of sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.025% bromphenol blue), boiled for 5 min, and stored at −20 °C for at least 30 min. For histone purification, the acid-soluble histone fraction of bone marrow was precipitated with acetone (32, 33) and dissolved in H2O.
Proteins were separated on 15% polyacrylamide gels and stained with 1% Coomassie Brilliant Blue in 50% methanol, 10% acetic acid. Triton-acetic acid-urea (TAU) gels (15% acrylamide) were run according to Waterborg (34). Densitometry was carried out using ImageMaster (Amersham Biosciences).
Bone marrow proteins (100 μg) were separated by non-equilibrating pH gradient gel electrophoresis (35) in 15-cm rod gels containing carrier ampholytes (pH 3–10) for 2400 V-h. The gels were equilibrated for 10 min in 2% SDS, 5% dithiothreitol, 10% glycerol, trace bromphenol blue, and 1.5% Tris-HCl, pH 6.8 (36) prior to SDS-PAGE in 15% polyacrylamide gels. Proteins were visualized by silver staining (37). Four identical two-dimensional gels were run for the 14C-containing bone marrow, and four gels were run for the controls. Each of the labeled gels and three unlabeled gels were analyzed separately by AMS. A fourth control gel was used for in-gel digestion, peptide extraction, and identification by MALDI-MS and LC-ESI-MS.
Reversed phase HPLC was performed on a Beckman Gold system. Core histones were separated based on previously published methods (32, 33) using a 180-μm × 15-cm C4 column (Vydac) and eluted with a 38–55% B linear gradient over 120 min (Buffer A: 0.1% heptafluorobutyric acid in H2O; buffer B, 0.08% heptafluorobutyric acid in acetonitrile). HPLC separation of AspN-digested peptides of histone H4 was carried out using a C18 column (180 μm × 15 cm) (LC Packings, San Francisco, CA) with an Applied Biosystems 140B syringe pump.
Disposable materials were used for any item that might come in contact with a sample to prevent cross-contamination between samples. Gel spots were excised with disposable plastic drinking straws and placed in 6- × 50-mm quartz tubes. Tributyrin was added as a carrier to HPLC fractions. Samples were dried by vacuum centrifugation, and the dried samples were reduced to graphite using published protocols (38). For [14C]benzene analysis, we measured 14C relative to 13C and normalized to the ratio of 1950 AD carbon using the Australian National University sugar reference standard (39). The carbon contents of the samples was determined using a C:N:S analyzer (Carlo-Erba NA1500, series 2). Hemoglobin was determined to have a carbon content of 50.38%.
The procedure of Clauser et al. (40) was used with minor modifications for the tryptic digestion of proteins in polyacrylamide gels. Each spot was excised, diced, and vortexed three times for 10 min in 50% acetonitrile, 25 mm ammonium bicarbonate. The gel pieces were dried in a Speedvac, resuspended in 25 mm ammonium bicarbonate with 12.5 ng/μl trypsin, and digested overnight at 37 °C. The peptides were extracted by vortexing three times for 10 min in 50% acetonitrile, 5% trifluoroacetic acid. Extracts were pooled, concentrated in a Speedvac to ∼1–2 μl, and dissolved in 10 μl of 50% acetonitrile, 5% trifluoroacetic acid. HPLC-separated histone H4 was digested with AspN for 4 h in 50 mm sodium phosphate, pH 8.0 at 37 °C.
The molecular masses of peptides were determined by analyzing the unseparated tryptic digests using one of three mass spectrometric techniques. Routine peptide analysis utilized a MALDI-TOF DE STR mass spectrometer (Applied Biosystems, Framingham, MA) operated in reflectron mode. One-tenth of each sample digest was mixed in a 1:1 ratio with 33 mm α-cyano-4-hydroxycinnamic acid solution (Agilent, Palo Alto, CA). Spectra were internally calibrated using known trypsin autolysis products (41). Selected ions were subjected to postsource decay analysis (42, 43). High energy positive ion collision-induced dissociation mass spectra were acquired on a Kratos Analytical Instruments Concept IIHH four-sector tandem mass spectrometer (Kratos, Manchester, UK) (44) equipped with a continuous flow, liquid inlet probe for liquid secondary ionization and a scanning charge-coupled device array detector. For HPLC-MS, a capillary HPLC system (ABI, Foster City, CA) was interfaced directly to a Mariner orthogonal acceleration time-of-flight mass spectrometer using electrospray ionization (Applied Biosystems). The unseparated digests were diluted in water and injected directly into a 180-μm × 15-cm capillary C18 column (LC Packings). Online HPLC-MS was performed using a formic acid/ethanol/propanol solvent system (45) with a gradient of 5–60% solvent B in 2 h. A cone voltage setting of 175 V was used for collision-induced dissociation, giving rise to fragment ion peaks. Peptide masses and fragment ion masses measured by either MALDI-MS or LC-ESI-MS were analyzed with the MS-Fit and MS-Tag programs in ProteinProspector (prospector.ucsf.edu) (46). Mass accuracy in both MALDI-MS (internally calibrated) and ESI-MS was generally 50 ppm or better.
AMS was used to determine the 14C incorporation in proteins separated by one-dimensional SDS-PAGE with Coomassie Blue staining throughout the molecular mass range up to 120 kDa in liver (low dose only) and bone marrow (low dose and high dose). In the liver, the highest levels of the heavy isotope were found in the protein fraction corresponding to the molecular mass range of 45–55 kDa (Fig. 1, upper panel). By contrast, in bone marrow the distribution of 14C proteins for both low and high dose showed the highest levels of incorporation in a band of 12–15 kDa. The distribution of 14C throughout the molecular mass range was similar for both doses of benzene in the bone marrow. It was noted that all regions of the gels showed significant levels of isotope incorporation compared with naturally occurring levels in undosed controls, suggesting that at least a substantial fraction of this incorporation was nonspecific. We performed similar gel analysis on fractions enriched for cytosolic, nuclear, and microsomal proteins from exposed liver and bone marrow to obtain subcellular localization for protein targets of benzene metabolites. The highest levels of incorporation in the liver were found in microsomes and cytosol, whereas the bone marrow showed 14C enrichment in the nuclei (data not shown).
14C content in gel slices cut from SDS-PAGE-separated proteins from liver (low dose) and bone marrow (low dose and high dose). Each gel lane was divided into sections and submitted for AMS analysis. The data are expressed as the percentage of total 14C incorporation across each gel lane after subtraction of controls. For the upper two panels the controls were sections of gel between the lanes. For the lower panel the control was a gel lane run with protein from untreated mice. The left-hand side of each panel represents the top of the gel, containing proteins that did not enter the gel, typically of molecular mass >120 kDa. The right-hand side represents the dye front with proteins <10 kDa.
The protein fractions containing the highest heavy isotope levels corresponding to 45–55 kDa from liver and 12–15 kDa from bone marrow were subjected to in-gel trypsin digestion. The extracted peptides were separated by HPLC, their molecular masses were determined by MALDI-MS, and in some cases they were identified by tandem mass spectrometry using high energy collision-induced dissociation. Five different polypeptides were identified in the 45–55-kDa liver fraction: the α and β chains of ATP synthase, disulfide isomerase, aldehyde dehydrogenase, and elongation factor EF-1 α-chain (Table I). In the 12–15-kDa bone marrow fraction, we identified α- and β-hemoglobin and the core histones H2A, H2B, and H3 as unresolved components. Histones H2A, H2B, and H3 were also identified from the 12–15-kDa region of bone marrow fractions enriched for nuclear proteins.
The amount of 14C in each region of the gels was compared with relative protein abundances as determined by densitometry. It was noted that the profiles for isotope incorporation along the gel bands were very similar to the densitometer profiles, e.g. see the densitometer and 14C profiles for bone marrow (high dose) depleted in red blood cells (Fig. 2). This observation that 14C levels are proportional to protein concentration, independent of protein molecular mass, suggests that a large part of protein adduct formation is a nonspecific effect that is independent of the nature of the proteins.
Comparison of the density of staining (top) and the 14C content (bottom) in slices from a one-dimensional gel used to separate bone marrow proteins (high dose) depleted in red blood cells.Error bars represent the uncertainty in the AMS measurements. The left-hand side represents the top of the gel.
As histones had been identified in the one-dimensional gel slices with elevated 14C levels and as histones are central to the structure and function of chromatin, we surmised that molecular damage to histone proteins might be capable of producing the cellular abnormalities observed in benzene toxicity. It was desired to establish whether histones were indeed a target for benzene metabolite modification. Because the histone and hemoglobin signals were coincident in the same one-dimensional slices, we were concerned that higher levels of enrichment in low level proteins could be masked by more abundant species, therefore two-dimensional separation was carried out. Histone proteins have basic isoelectric points in the range of 10–11.5; consequently the cellular homogenate was separated in the first dimension using non-equilibrating pH gradient gel electrophoresis, which is optimal for the separation of basic polypeptides (32). Four control gels of protein from mice not treated with benzene were prepared in an identical manner, one of which is shown in Fig. 3. Focusing primarily on the 12–15-kDa molecular mass and basic pI range, selected spots were excised for AMS (four treated and three untreated gels) or conventional mass spectrometry (one untreated gel). Other more intensely stained spots corresponding to proteins of higher molecular mass were also taken for analysis.
One of four silver-stained two-dimensional gels from electrophoresis of protein homogenates from bone marrow extracted from untreated mice. Four identical gels were prepared from bone marrow extracted from mice treated with benzene (high dose). The numbered spots were cut from all the treated gels and three of the untreated gels and subjected to 14C analysis by AMS. Equivalent spots from the remaining untreated gel were used for protein identification by conventional mass spectrometry.
The gel spots intended for conventional mass spectrometry were subjected to in-gel digestion and identification by MALDI-MS and/or LC-ESI-MS, the results of which are summarized in Table II. The most strongly stained spots, 21 and 22, were found to include both α- and β-hemoglobin. Spots 30 and 31 at ∼30 kDa also gave peptide masses matching both α- and β-hemoglobin, most likely due to hemoglobin dimers. Spots 27 and 28 in the ∼10-kDa region of the gel also contained α- and β-hemoglobin, N-terminal and C-terminal peptides being seen for both isoforms. As these do not appear to be truncated forms of the protein, their presence in this region of the gel is surprising and unexplained. Of the remaining highly basic spots in the ∼12–14-kDa region, 23 and 33 were identified as containing a mixture of histone H2A and histone H2B, whereas spot 35 was identified to contain only histone H2B. Spot 25 was identified as the murine cytokine CP-10, the peptides giving 87% sequence coverage. No identifications could be made for spectra from in-gel digests of spots 24, 34, 36, and 37.
Due to the polymorphism of β-hemoglobin in mouse, several variants were found. Each peptide is marked as belonging to one of the following variants: s (single), j (D major), or n (D minor).
The AMS measurements shown in Fig. 4 for the excised gel spots demonstrate that the strongly stained spots 21 and 22 contained the bulk of the isotopic label, and the weakly stained histone spots showed only low levels of incorporation. Silver staining is not ideal for quantitation, particularly with the wide dynamic range of proteins represented by these gels, and the basic histones are known to stain poorly with silver. Therefore from this experiment it was not possible to accurately compare the relative amounts of incorporation of 14C into the histones per unit of protein. However, compared with hemoglobin, these data gave no indication of any low level proteins being significantly more enriched in 14C.
Mean incorporation of 14C in bone marrow proteins in spots cut from the two-dimensional gels.Error bars show standard deviations from four experiments using benzene-treated mice (solid bars) and three using untreated controls (open bars). The inset shows a 3× expanded view of all but the two strongest signals.
The foregoing experiments suggested that the histones were not necessarily specific targets for higher than average incorporation of benzene metabolites. However, not all the histones had been identified by the two-dimensional gel separation. Therefore, in a separate experiment, core histones were purified from the bone marrow of mice receiving the high dose of [14C]benzene. The histones were separated on one-dimensional TAU gels and stained with Coomassie Brilliant Blue, which would give more quantitative staining. The heavy isotope content was determined by AMS, and the proteins in each band were identified by trypsin digestion and MALDI mass spectrometry. Fig. 5 shows one lane from a TAU gel, the histones identified within each band, and the 14C content of each band as determined by AMS. The wide dynamic range for the stained bands that were identified as containing histones is reflected in the widely varying 14C content. However, when the AMS results were normalized based on the protein abundances as quantitated by densitometry, all identified histones, H1, H2A, H2B, H3, and H4, contained the same amount of the isotopic label per unit of protein within ±30%. This experiment clearly shows that the degree of 14C incorporation is independent of the specific histone, and no histones stand out as being particularly susceptible to covalent modification by benzene or its metabolites.
Incorporation of 14C in core histones separated using TAU gels.Left, TAU gel with histones identified by MALDI; bands labeled * were not identified. Right, 14C incorporation measured by AMS, expressed as fraction modern (38).
A representative histone was chosen for further study to determine whether or not the covalent modification by benzene metabolites was associated with specific amino acids or sites within the protein. HPLC-purified histone H4 from the high dose experiment was digested in solution using endoproteinase AspN, the peptides were separated by HPLC, and fractions were collected (Fig. 6, upper panel). One-tenth of each fraction was analyzed by conventional mass spectrometry to identify the peptides (Table III), and the remainder of each fraction was analyzed for 14C content by AMS as shown by the gray bars in the lower panel of Fig. 6. Full coverage of the protein sequence was not obtained; some fractions were found to contain multiple peptides, and no peptides were identified in the later fractions. Although the levels of 14C determined by AMS showed wide variation across the various fractions, when the level of 14C incorporation was normalized to the area of the HPLC peaks, which for absorption at 214 nm should be approximately proportional to the product of peptide concentration and the number of amino acids per peptide, all HPLC fractions showed significant incorporation of the label, and there were no obvious peptides that were strongly favored for modification as shown by the black bars in Fig. 6.
Analysis of histone H4 digested with AspN after isolation from bone marrow (high dose).Top, reversed phase HPLC of peptides, monitored by UV absorption at 214 nm, showing fractions collected for peptide identification by conventional mass spectrometry (10% of each fraction) and measurement of 14C by AMS (90%). Bottom, gray bars show the amount of 14C detected in each fraction by AMS; black bars show the same quantity divided by the area of the corresponding HPLC peak (arbitrary units).
Note that several peptides resulted from nonspecific cleavages.
In this study we detected and measured covalent protein modifications by 14C-containing metabolites of benzene in the bone marrow of mice. This approach allowed us to measure attomole levels of isotope, in this case 14C, in individual proteins at levels of one adduct per 106 protein molecules. For β-hemoglobin (spot 22), we calculated an adduct level less than 1 fmol/μg. Using gas chromatography/mass spectrometry, hemoglobin had previously been identified as a target for modification by benzene metabolites (28, 29). However, the methodology developed here is applicable to the more general identification and characterization of proteins modified by xenobiotics in vivo at very low exposure levels and using small amounts of protein without prior knowledge of the structural nature of the adduct.
We note that a limitation of the AMS approach using 14C as a tracer is that it can only detect the presence of carbon-containing adducts. The methods adopted here would not detect other covalent changes arising from processes such as oxidation or hydrolysis of the targets.
The profile of 14C distributed across the one-dimensional gel slices revealed a differential distribution in the liver and bone marrow. However, the protein distributions for these samples were also different, and proteins identified in the same molecular mass range in the bone marrow and in the liver were not all the same. Hemoglobin was identified only in the 12–15-kDa bone marrow gel slice and not in the liver gel slice in the same molecular mass range. There could be numerous proteins in that molecular mass range of a one-dimensional gel slice, and it is likely that only a subset of these would be identified by mass spectrometry. Subcellular fractionation prior to one-dimensional analysis improved the dynamic range and simplified the identification of 14C-labeled proteins but still did not allow definitive identification of individual protein targets for modification.
To correlate heavy isotope incorporation with individual polypeptides, we used the high resolving power of two-dimensional electrophoresis to separate the components of the 12–15-kDa bone marrow band prior to AMS. The isotope label in the 12–15-kDa band of the bone marrow was partitioned primarily into the nuclear compartment, and we focused on the molecular mass and pI range in two-dimensional gels that would incorporate the histones to identify benzene adducts. The use of two-dimensional electrophoresis significantly improved the dynamic range over one-dimensional gels, and the use of in-gel digestion and mass spectrometric analysis permitted rapid identification of the proteins of interest from the two-dimensional gels. The majority of the label in the protein homogenates from bone marrow proved to be associated with hemoglobin. This likely resulted from peripheral blood in the bone marrow preparation. For future studies, contaminating mature red blood cells could be removed from bone marrow samples by suspending the sample in a hypotonic solution to lyse the red blood cells prior to homogenization (47).
The one-dimensional gel analysis suggested that histones might be targets for attack by benzene or its metabolites. This was confirmed by two-dimensional separation, although silver staining did not allow the quantitation of the modified species. However, subsequent enrichment of the histones and one-dimensional separation by TAU gels with more quantitative Coomassie Blue staining gave no indication that any specific histone was a favored target for modification. This is despite the fact that the amino acid compositions of the various histones vary quite widely. For example histone H2A contains one cysteine residue that might be a target for modification, whereas H2B contains no cysteine residues. All histones are lysine-rich, and the nucleophilic ϵ-amino group of lysine may also be a binding site for benzene metabolites. Few peptides derived by digestion of H4 contain no lysine residues, and every HPLC fraction was found to contain at least one peptide that included at least one lysine residue. Thus the observation that all HPLC fractions gave similar 14C levels when normalized to the area of their UV peaks at 214 nm suggests that their susceptibility to modification by reactive benzene metabolites could be due to the presence of lysine, although it should be noted that alanine, arginine, glycine, isoleucine, leucine, threonine, and tyrosine were also represented in at least one peptide within each fraction.
The findings reported here demonstrate that benzene metabolites are highly reactive and appear relatively promiscuous in their selection of protein targets. Detailed investigation of the histones demonstrates that their attack by reactive benzene species is ubiquitous, resulting in multiple modified sites within a single histone. Of the amino acids potentially susceptible to reaction, cysteine is not an exclusive target, whereas lysine could be. The evidence suggests that multiple lysine residues within each histone are attacked, the location of each modification being based on rates of diffusion rather than kinetic factors. This highly reactive nature of the benzene metabolites suggests a free radical mechanism. A number of authors have presented evidence for free radical mechanisms in benzene toxicity as reviewed by Subrahmanyam et al. (17). As free radicals the metabolites would have short half-lives, therefore protein targets for attack would need to be closely associated with a region in which benzene or its less reactive metabolites might accumulate after environmental or occupational exposure. Accumulation in the bone marrow cells could place the substrate for formation of reactive species close to chromatin, thereby allowing histone modification to proceed relatively efficiently. Accumulation in red blood cells would also contribute to the modifications seen in hemoglobin.
The modification of histone proteins by the known leukemogenic agent benzene is intriguing in light of the role of histones in transcription and chromosome structure and function. Acetylation of the highly conserved lysine residues in the N-terminal tails of the core histones results in chromatin structural changes and gene expression (48–51). Lysine acetylation reduces inhibition of transcription, and abnormal acetylation/deacetylation can lead to cancer development (52). A number of histone deacetylase inhibitors are under investigation as novel anticancer drugs (53). Histone methylation has also been known for a long time, and it is now realized that, like acetylation, methylation also plays an important role in biological regulation (54). We have recently described analytical methods that greatly increase the sensitivity with which specific sites of both acetylation and methylation can be identified in histones (55, 56). The acetylated histones have a lower affinity for chromatin and may be more accessible to modification. The turnover of histones is low (57), so the effects of modification by benzene metabolites could be stable and could yet prove to play a role in the process of carcinogenesis.
In earlier work on the identification of the hepatic protein targets of acetaminophen and a non-toxic regioisomer, we were able to use conventional tandem mass spectrometry not only to identify the major proteins but also to characterize the nature and sites of modification (58, 59). In the current study, any further structural analysis subsequent to AMS was hampered by small sample sizes and low stoichiometry, resulting from the apparently indiscriminate modification of a wide range of targets. Because there proved to be many proteins over a wide range of molecular masses susceptible to benzene modification, we could not identify the chemical structures of the reactive metabolites of benzene or the particular sites of modification. To achieve this it will be necessary to maximize the sensitivity of other mass spectrometric techniques to provide further information about the molecular changes associated with benzene exposure, including the site-specific structural characterization of the histone modifications indicated by the AMS analysis. Hopefully the identification of the structures of protein adducts would also identify the reactive species responsible and could indicate the metabolic pathways that are most important in benzene carcinogenicity.
In identifying histones as targets for benzene modification, this study validates a new method for identifying molecular modifications that will impact future biological studies. AMS detection of 14C, being up to 106-fold more sensitive than scintillation counting (60), allowed us to localize isotopically labeled adducts in one-dimensional and two-dimensional gel separations of proteins from mice exposed to low doses of [14C]benzene with attomole sensitivity. Proteins were then identified by established mass spectrometric methods. In the future this combination of high sensitivity detection of protein modification and high resolution separation and protein identification can be applied to study a wide range of trace toxic, carcinogenic, and therapeutic compounds at physiologically relevant levels and to monitor biological events occurring with low frequency.
We gratefully acknowledge F. Nilsson, K. Haach, V. Laiko, C. Mani, and K. Curtis for technical assistance and S. P. H. T. Freeman and B. A. Bucholz for operation of the accelerator mass spectrometer.
↵* This work was supported by University of California Systemwide Campus-Laboratory Consortium (to A. L. B.), National Center for Research Resources (NCRR) Grant RR13461 (to K. W. T.), NCRR Grant RR01614 (to A. L. B.), and Liver Center Grant DK26743 (to A. L. B.).
Revision received October 15, 2002.


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