Article 8

Science Debates Using

Tools to Redesign Life

Keith Schneider

WASHINGTON. June 7-Genetic engineering, the most powerful and precise biological tool for manipulating life ever devised, has reached a milestone.

Fourteen years after scientists first spliced genetic material from one microbe into another to create a bit of life that never before existed, genetic alterations once confined to science fiction are becoming ever more common.

Now the United States Patent and Trademark Office has ruled that genetic engineers may patent higher life forms-even mammals. The decision promises to widen vastly the commercial and agricultural applications of novel methods of producing new kinds of life.

Industrial leaders say they must be able to patent new life forms and processes if they are to protect their investments and move forward in a field full of innovation and risk. But the patent office ruling has also revived anxiety about the safety and morality of tampering with life forms.

That concern prompted a Congressional committee to schedule hearings this week on ethics and regulations in the field of genetic engineering. Last month, the Senate approved a measure that would prevent the Patent Office from spending money on reviewing patents for animals, but it still faces a conference committee vote.

In the near future biotechnology may see these developments:

In laboratories across the country, the genes of viruses and bacteria will be placed in plants to enable them to Produce their own insecticides or fertilizers. These so-called transgenic plants will be field-tested and farmers will begin using them in place conventional crop varieties.

* Researchers will manipulate the primordial cells that produce sperm and eggs to enable breeders to select the characteristics of animals, including gender.

* Scientists will routinely transplant genes from one species to another.

As the debate unfolds, many eyes will turn to a rust-colored pig in Beltsville, Md., with the growth hormone gene of a cow. That pig represents success to the genetic engineers and, because of its pathetic infirmities, new reason for concern to those who fear that mankind now has too many tools for meddling in the complex matter of life.

In recent months most of the concern about genetic engineering centered on the release into the environment of newly devised organisms in the form of bacteria designed to help plants resist pests, diseases and bad weather. With the new patent ruling, however, the concern has begun to shift to more complicated genetic manipulation of higher life forms-mammals-resulting in transgenic creatures like the pig with a cow gene.

In the long run, opponents and proponents of genetic engineering see a vast array of potential applications, including plants and microbes designed to produce fuel; cows that produce medicines instead of milk, or even babies destined to have a particular height, hair color or other traits.

The genetic traits of plants and animals have been manipulated for centuries. But until now animal breeding and the hybridization of crop plants have been slow, cumbersome and difficult. Furthermore, until now, breeders were never able to introduce genes from one species into another or to make such extensive changes.

Because many breeding techniques such as artificial insemination, in vitro fertilization and embryo transfer, have already made their way into medicine there are some who fear it may not be long until the manipulation of animal traits will extend to human traits as well.

"important legal, constitutional, and policy issues were raised by this decision," said Representative Robert W. Kastenmeier, a Wisconsin Democreat who heads the House Judiciary Sub- committee on Courts, Civil Liberties, and the Administration of Justice, which will hold the hearing Thursday on the Patent Office ruling.

The commercial applications of genetic engineering are already apparent. Sales of genetically engineered products, most of them new pharmaceuticals, have almost doubled annually in recent years and topped $350 million last year, according to industry analysts. The Congressional Office of Technology Assessment has identified almost 400 companies seeking to develop products with genetic engineering and other modern biological technologies. More than #3 billion, two-thirds of it provided by the Government, will be invested this year in biotechnology research, according to the General Accounting Office and industry analysis.

Yet as the ambitions and accomplisments of genetic engineering increase, awareness of its power and potential is generating a mixture of fascination and hope, aversion and misunderstanding.

A survey of 1273 American adults published in May be the Congressional technology office found that 2whil a majority of those interviewed believed that the potential benefits of genetic engineering outweighed its risks, they were disturbed by some applications, particularly the release of manufactured life forms into the environment and manipulations in human embryos.

2. GENETICS

"People understand at a gut level that there is something wondrous, and perhaps perilous, about a technology that changes the blueprint of life and will force us to make choices that are likely to be more profound than anything we, as a society, have ever faced:' said Senator Albert Gore Jr., a Tennessee Democrat who has studied the biotechnology industry.

Though scientists generally agree the field offers great promise, there is sharp disagreement over its potential perils.

"We are bringing a completely human-centered utilitarian attitude toward life:' said Dr. Michael Fox, a veterinarian and scientific director of the Humane Society of the United States. "All of earth's living things will simply become items to exploit'

Other scientists and many biotechnology industry executives insist that genetic manipulation will hasten the development of cures for diseases like AIDS, lead to solutions for toxic chemical pollution, produce a new agricultural cornucopia and open an industrial era based not on fossil fuels and chemicals, but on new, non-polluting substances produced by genetically engineered plants or microbes..

Genetic engineering was recognized as a momentous development in 1973, when Stanley N. Cohen of Stanford University and Herbert W. Boyer of the University of California at San Francisco snipped a piece of the genetic code out of one bacterium and inserted it into another.

But that experiment was followed almost immediately by a host of safety and ethical questions, many of which remain unresolved. Are living, genealtered microbes safe to release outdoors? What is the best way to assess the risk from such uses? Is it ethical to after the genetic codes of animals? What about people? How can a society know whether a new technology should be pursued or ignored?

"The issues range from ethics within universities, to the environment, to eugenics, to definitions of nature, to religious thought, to what it is to be human:' said Dorothy Nelkin, a professor in Cornell University's Program on Science, Technology and Society. "Other disputes over technology have been much simpler and mostly focus on health concerns."

The source of the and the excitement and the conflicts is a technique, conceptually simple but in practice quite complex. for rearranging basic hereditary material: deoxyribonucleic acid, or DNA that makes up genes.

DNA molecules are long, twisted ladders of chemicals called nucleotide bases: adenine, thymine, guanine and cytosine. More than 30 years ago, scientists determined that adenine always pairs with thymine, and cytosine with guanine. These chemical connections are called base pairs; a single gene, a section of DNA, is typically made up to 10,000 to 20,000 base pairs. Human beings, it is estimated, have between 100,000 and 200,000 genes, or up to 4 billion base pairs, organized on 46 chromosomes.

Though the numbers of genes in mammals, plants, and microbes differ, their ladder-like molecular structure does not. Scientists are now able to identify and isolate specific genes and remove them with proteins, called restriction enzymes, that slice DNA in specific places. The enzymes cause the pairs on either end of the gene to split, leaving nucleotide bases without corresponding mates. Scientists paired bases "sticky ends' seeking the correct chemical fit, they easily merge with another organism's genetic structure.

Yet simply isolating a gene from one animal and plugging it into another does not mean that the gene will produce the desired result. A gene's functions are determined by its location on a chromosome, the workings of neighboring genes and other factors that are still mysteries.

So far, genetic engineers are largely limited to transferring single genes into microbes, plants and animals, or taking single genes out of bacteria and viruses. Alterations involving more than one gene, such as creating crops that produce their own insecticides and fertilizer, or cows that produce medications in their udders instead of milk, are still years away.

Assertions that genetic engineering will produce unrecognizable plants or monstrous animals are considered by many researchers to be scientifically absurd.

"There are severe limits to the extent of the modifications we can make:' said Dr. Bernard D. Davis, a microbiologist at Harvard Medical School. "if you mix genes from genetically distant organisms that don't fit each other well, you will not have an organism that can live."

We're not gong to make weeds out of non-weedy species said Dr. Winston

Brill, vice of research and development at Agracetus, a plant biotechnology company in Middleton, Wis. "Were not going to have Frankensteins crawling around. "

Nevertheless, transfers involving a single gene can yield striking physiological changes.

For example, the transgenic pig, a rust-colored boar born last November at the Department of Agriculture's experiment station in Beltsville, now weighs as much as its natural cousins; but unlike them little of its bulk is fat. But it has trouble walking on short legs swollen by arthritis. Its eyes, peering from a broad and wrinkled face, are slightly crossed. If it is like its father, who was one of the world's first transgenic farm animals, it will not live to be two years old.

Nothing about producing transgenic animals is easy. Genes are injected into fertilized animal eggs. Piercing cell walls kills between half and three quarters of the eggs, said Dr. Vernon G. Pursel, the research physiologist conducting the swine experiments. In four years, scientists injected more than 8,000 fertilized eggs to produce just 43 transgenic Pigs.

It is little wonder, then, that researchers at Beltsville consider the birth of the rust-colored pig to be a scientific success. The young boar inherited the gene that scientists inserted into its father, and the gene expressed itself. Scientists are now working to control the gene so that it produces animals that grow fast, eat less, and produce more lean meat, without the complex of crippling diseases afflicting the boar.

The Foundation on Economic Trends, a small public policy group that opposes genetic engineering, and the Humane Society of the United States unsuccessfully filed suit in Federal District Court three years ago to halt the research that produced the rust-colored boar's father. They said the research was cruel, violated the innate dignity of animals and would have significant social and economic effects by producing bigger, more expensive animals that would cause dislocations in the farm economy.

"That kind of scientific reductionism undermines the respect for life and future generations will come to regret it:' James Rifkin, president of the Foundation, said recently.

The two groups are also protesting the new Patent Office policy. In this battle they are joined by farm organizations, consumer groups, environmental groups and most major animal welfare groups-

Famers believe they will be facing fewer choices in terms of breeds avail-

Able to them and will be paying far more for animals." Said R. Keith Stroup, legal counsel for the League of Rural Voters,

43 Years of Advances

in Altered Life Forms

Keith Schneider

WASHINGTON, June 7- The advances that make genetic engineering possible began in 1944, when Oswald T. Avery, Colin MacLeod and Maclyn McCarthy, researchers at Rockefeller University in New York, determined that deoxyribonucleic acid, or DNA, carried the hereditary blueprint of all living things. The same year, Congress passed the Public Health Service Act, which provided grants to universities for medical and biological research.

The work at Rockefeller University and the new funds for exploring its implications ignited a field where the pace of discoveries, slow at first, has accelerated to the point at which important findings are being announced almost weekly.

A major riddle was solved in 1953, when James D. Watson and Francis H. Crick found that DNA was chemically organized in two strands, a double helix. Their findings, which earned them a Nobel prize in 1962, enabled researchers to understand how DNA worked. By the mid-1960's, biochemists and molecular biologists were able to purify fragments of DNA, tag the molecules with radioactive isotopes and analyze the fragments.

In 1970, scientists developed proteins, or restriction enzymes, that cut DNA strands in precise locations. Three years later, Stanley N. Cohen of Stanford University and Herbert WBoyer of the University of California at San Francisco used the restriction enzymes to isolate fragments of DNA in one bacterium and insert R into another. Genetic engineering was born.

Scientists reacted quickly. In September 1973, and again in July 1974, several of the country's leading biologists warned in eminent journals that the new gene-splicing techniques might present novel hazards. They asked scientists to delay research until the dangers of genetic engineering could be more carefully evaluated.

Three months later the National Institutes of Health established a committee to monitor biotechnology research and to consider new research rules; the following February, 139 scientists from 19 nations met in California to draw up new research guidelines to minimize the potential that gene-altered microbes could escape from laboratories. The guidelines were adopted by the N.I.H. in 1976.

By then, investors and industrialists had seen the commercial possibilities of the new field. In i976, Genentech in South San Francisco, Calif., became the first company established to commercialize the new technology.

In the early 1980's, pharmaceutical makers, chemical companies and other concerns signed university researchers to long-term contracts aimed at quickening the pace of research with commercial potential. Some scientists and scholars cautioned that such relationships could alter the direction of research and stifle the free exchange of scientific information. But the lucrative contracts proved worthwhile for many universities, companies and scientists.

The first important court decision on genetic engineering came in 1980, when the United @ Supreme Courtoverturning a policy of the Patent and Trademark Office, ruled 5 to 4 that an oiwating microbe developed by a General Electric researcher could be patented. Bolstered by this ruling, the pace of discovery quickened.

In 1981, the Food and Drug Administration approved an application by Eli Lilly & Company to market the first drug made from genetically altered bacteria, a form of insulin.

On Jan. 16,1986, the Department of Agriculture granted the Biologics Corporation of Omaha, Neb., the world's first license to market a living, genetically engineered microorganism, a virus used as a vaccine to prevent a herpes disease in swine. It was tested in Lometa, Tex., in June 1984.

On May 29,1986, Agracetus, a biotechnology company in Wisconsin, was the first to field test a genetically engineered crop plant, a genetically altered form of tobacco it planted at an undisclosed site 20 miles from its headquarters.

On June 19,1986 President Reagan signed into law a coordinated set of rules and regulations governing the testing, use and sale of the products of biotechnology. Though some scientists have said some sections of the law are based on incorrect theories, the program is serving as a model for biotechnology regulation in other countries.

On April 16, the United States Patent and Trademark Office decided to extend patent protection from microbes to higher life forms-even mammals.

And on April 24, 1987, after four years of delays because of legal challenges by Jeremy Rifkin, a public policy activist involved in biotechnology issues, the first free release of genetically engineered bacteria was conducted outside Brentwood, Calif.

A family farm advocacy group that opposes the policy. "There's another issue here, too. Most farmers who deal with animals on a day-to-day basis want to be very thoughtful and careful about tampering with life. Clearly we have not explored fully the repercussions, morally and ethically, of what these Patent applications seek to do."

Fifteen applications have been filed, but the office does not release descriptions of patent proposals until they are approved.

Dr. Pursell said he was sensitive to the protests but unsure how to respond. We are not doing anything out here that is cruel" he said, adding, "The research could have a tremendous practical value."

Other applications of genetic engineering technology are less invasive and also less divisive. Scientists have discovered several methods for moving bacterial and viral genes into plants to

Major Personality Study

Finds That Traits

Are Mostly Inherited

Daniel Goleman

The genetic makeup of a child is a stronger influence on personality than child rearing, according to the first study to examine identical twins reared in different families. The findings shatter a widespread belief among experts and laymen alike in the primacy of family influence and are sure to engender fierce debate.

The findings are the first major results to emerge from a long-term project at the University of Minnesota. Since 1979, more than 350 pairs of twins in the project have gone through six days of extensive testing that has included analysis of blood, brain waves, intelligence and allergies.

The results on personality are being reviewed for publication by the Journal of Personality and Social Psychology. Although there has been wide press coverage of pairs of twins reared apart who met for the first time in the course of the study, the personality results are the first significant scientific data to be announced.

For most of the traits measured, more than half the variation was found to be due to heredity, leaving less than half determined by the influence of parents, home environment and other experiences in life.

The Minnesota findings stand in sharp contradiction to standard wisdom on nature versus nurture in forming adult personality. Virtually all major theories since Freud have given far more importance to environment, or nurture, than to genes, or nature.

Even though the findings point to the strong influence of heredity. the family still shapes the broad suggestion of personality offered by heredity; for example, a family might tend to make an innately timid child either more timid or less so. But the inference from this study is that the family would be unlikely to make the child brave. The 350 pairs of twins studied included some who were raised apart. Among these separately reared twins were 44 pairs of identical twins and 21 pairs of fraternal

twins. Comparing twins raised separately with those raised in the same home allows researchers to determine the relative importance of heredity and of environment in their development. Although some twins go out of their way to emphasize differences between them in, general identical twins are very much alike in personality.

"If in fact twins reared apart are that similar, this study is extremely important for understanding how personality is shaped:' commented Jerome Kagan, a developmental psychologist at Harvard University. "It implies that some aspects of personality are under a great degree of genetic control."

The traits were measured using a personality questionnaire developed by Auke Tellegen, a psychologist at the University of Minnesota who was one of the principal researchers. The questionnaire assesses many major aspects of personality, including aggressiveness, striving for achievement, and the need for personal intimacy.

For example, agreement with the statement "When I work with others, I like to take charge" is an indication of the trait called social potency, or leadership, while agreement with the sentence "I often keep working on a problem, even if I am very tired" indicates the need for achievement.

Among traits found most strongly determined by heredity were leadership and, surprisingly, traditionalism or obedience to authority. "One would not expect the tendency to believe in traditional values and the strict enforcement of rules to be more an inherited than learned trait:' said David Lykken, a psychologist in the Minnesota project. "But we found that, in some mysterious way it is one of the traits.with the strongest genetic influence."

Other traits that the study concludes were more than 50 percent determined by heredity included a sense of well-being and zest for life; alienation; vulnerability or resistance to stress; and fearfulness or risk seeking.

Another highly inherited trait, though one not commonly thought of as part of personality, was the capacity for becoming rapt in an aesthetic experience, such as a

concert.

Vulnerability to stress, as measured on the Tellegen tests what is commonly thought of as "neuroticism, "according to Dr. Lykken. "People high in this trait are nervous and jumpy, easily irritated, highly sensifive to stimuli, and generally dissatisfied with themselves, while those low on the trait are resilient and see themselves in a positive light:' he said. "Therapy may help vulnerable people to some extent, but they seem to have a built-in susceptibility that may mean, in general, they would be more content with a life low in stress."

The need to achieve, including ambition and an inclination to work hard toward goals, also was found to be genetically influenced, but more than half of this trait seemed determined by life experience. The same lower degree of hereditary influence was found for impulsiveness and its opposite, caution.

The need for personal intimacy appeared the least determined by heredity among the traits tested; about two-thirds of that tendency was found to depend on experience. People high in this trait have a strong desire for emotionally intense relationships; those low in the trait tend to be loners who keep their relationship troubles to themselves.

"This is one trait that can be greatly strengthened by the quality of interactions in a family," Dr. Lykken said. "The more physical and emotional intimacy, the more likely this trait will be developed in children, and those children with the strongest inherited tendency will have the greatest need for social closeness as adults."

No-single gene is believed responsible for any one of these traits. Instead, each trait, the Minnesota researchers propose, is determined by a great number of genes in combination, so that the pattern of inheritance is complex and indirect..

No one believes, for instance, that there is a single gene for timidity but rather a host of genetic influences. That explain, they say, why previous studies have found little connection between the personality traits of parents and their children." Whereas identical twins would share with each other the whole constellation of genes that might be responsible for a particular trait, children might share only some part

of that constellation with each parent

'That is why, just as a short parent may have a tall child, an achievement -oriented parent might have a child with little ambition.

The..Minnesota findings are sure to stir debate. Though most social scientists accept the careful study of twins, particularly when it includes identical twins reared apart, as the best method of assessing the degree to which a trait is inherited, some object to using these methods for assessing the genetic component of complex behavior patterns or question The conclusions that are drawn from it.

Years ago, when the field was dominated by a psychodynamic view, you could not publish a study like this, Dr. Kagan Added "Now the field is shifting to a greater acceptance of genetic determinants, and there is the danger of being too uncritical of such results."

Seymour Epstein, a personality psychologist at the University of Massachusetts, said he was skeptical of precise estimates of heritability. "The study compared people from a relatively narrow range of cultures and environments:' he said. "If the range had been much greater-say Pygmies and Eskimos as well as middle-class Americans-then environment would certainly contribute more to personality. The results might have shown environment to be a far more powerful influence than heredity," he said.

Dr. Tellegen himself said: "Even though the differences between families do not account for much of the unique attributes of their children, a family still exercises important influence. In cases of extreme deprivation or abuse, for instance, the family would have a much larger impact-though a negative one-than any found in the study. Although the twins studied came from widely different environments, there were no extremely deprived families' "

Gardner Lindzey, director of the Center for Advanced Studies in the Behavioral Sciences in Palo Alto, Calif., said the Minnesota findings would "no doubt produce empassioned rejoinders."

"They do not in and of themselves say what makes a given character trait emerge:' he said, "and they can be disputed and argued about, as have similar studies of intelligence."

For parents, the study points to the importance of treating each child in accord with his innate temperament.

"The message for parents is not that it does not matter how they treat their children, but that it is a big mistake to treat all kids the same:' said Dr. Lykken. "To guide and shape a child you have to respect his individuality, adapt to it and cultivate those qualities that will help him in life."

"If there are two brothers in the same family, one fearless and the other timid, a good parent will help the timid one become less so by giving him experiences Of doing well at risk-taking, and let the other develop his fearlessness tempered with some intelligent caution. But if the parent shelters the one who is naturally timid, he will likely become more so."

The Minnesota results lend weight and precision to earlier work that pdifited to the importance of a child's temperament in development. For instance, the New York Longitudinal Study, conducted by Alexander Thomas and Stella Chess, psychiatrists at New York University Medical Center, identified three basic temperaments in children, each of which could lead to behavioral problems if not handled well.

"Good parenting now must be seen in terms of meeting the special needs of a child's temperament, including dealing with whatever conflicts it creates," said Stanley Grossman, a staff member of the medical center's Psychoanalytic Institute.

The New York Times. Dec 1. 1986

The Roots of Personality

The degree to which eleven key traits of personality are estimated to be inherited. as gauged by tests with twins. Traits were measured by the multidimensional Personality Questionnaire, developed by Auke Tellegen at the University of Minnesota.

SOCIAL POTENCY 61%

A person high in this trait is masterful. a forceful leader who likes to be the center of attention.

TRADITIONALISM 60%

Follows rules and authority, endorses high moral standards and strict discipline.

STRESS REACTION 55%

Feels vulnerable and sensitive and is given to worries and easily upset.

ABSORPTION 55%

Has a vivid imagination readily captured by rich experience. relinquishes sense of reality.

ALIENATION 55%

Feels mistreated and used. that "the world is out to get me."

WELL-BEING 54%

Has a cheerful disposition, feels confident and optimistic

HARM AVOIDANCE 51%

Shuns the excitement of risk and danger, prefers the safe route even if it is tedious

"Is physically aggressive and vindictive, has taste for violence and is out to get the world".

"Works hard, strives for mastery and puts work and accomplishment ahead of other things."

Is cautious and plodding, rational and sensible, likes carefully planned events.

Prefers emotional intimacy and close ties, turns to others for comfort and help.

Philip Hilts

There is a drawer called the catastrophe file" in the office of biologist Charles Ehret at the Argonne National Laboratory. It holds reports of various disasters: an air crash that killed hundreds, a ship collision in which crew members drowned, a hospital accident that will bring members of the staff to trial, embarrassing errors in the Pioneer space probes, blunders in Middle East diplomacy.

During the first 100 minutes of the nuclear accident, the control room workers made a surprising series of mistakes. Fourteen seconds into the accident, one controller failed to see two warning lights. A few seconds later, a valve that should have closed did not, but operators did not realize it. As the president's investigating commission later said, "Throughout the first two hours of the accident, the operators ignored or failed to recognize the significance of several things that should have warned them that they had an open valve and a loss-of-coolant accident...." The president's commission concluded that' . except for human failures, the major accident at Three Mile Island would have been a minor incident."

One of the relationships discovered by chronobiology is that the many rhythms within the human body normally move in a set synchrony with one another. For example, body temperature, pulse, and sleep-wake cycles follow roughly the same beat, while other processes vary with a quite different beat-and the relations among them may change, slightly but predictably, day after day. Each gland, each organ, each chemical has its own beat, and together they are orchestrated as harmoniously as the players, in a symphony.

We think of our heartbeat a constant, but it is not. It will va@y as much as 20 or 30 beats per minute in 24 hours. At one time of day it may beat 60 times per minute, and at the opposite time of the circadian cycle, it may beat 80 or more times per minute. Blood pressure may measure 120 over 80 in the morning, but in the evening it is more likely to be higher, possibly as much as 140 over 100, an unusually high reading. Body temperature does not hover around 98.6 degrees, as most of us believe, but varies by one and a half to two degrees over a day, from as low as 97 degrees to more than 99 degrees. The scores of other functions which have been shown to swing up and down widely during the day include more than three dozen separate chemicals in the blood and urine, as well as mood, vigor, eye-hand coordination,,counting, time estimation, addition, and memory. Cell division rate in the body has been found to vary by 1,200 percent over a day; one chemical in the pineal gland varies by 900 percent over a day.

After the TMI nuclear accident, several utilities, including the General Public Utilities Corporation which runs the plant, turned to biologists for help in lessening the dangers of erratic human performance.

"We may be able to improve the situation by several orders of magnitude:' says Ehret, who has consulted with the nuclear utilities and drawn up alternate night work plans, all according to biologically based rules that have emerged from research in chronobiology.

But there is another, equally critical area where a failure to take into account the circadian fluctuations of the body's natural rhythms will lead to trouble: medicine and medical research.

In medicine, the prevailing biological idea has been that the body seeks equilibrium, a steady state. When it is ill, say with a fever, it tries to return to wellness by sweating and other means of cooling itself. This is the homeostatic view. It states that in each bodily function there is an ideal mean, and that a healthy person's functions will flutter randomly about that middle number. For example, in temperature, 98.6 degrees is the accepted mean. In blood pressure, 120 over '8'0 is the rough center.

Now it is clear that this approach is faulty, or at least incomplete. Bodily functions do fluctuate, but not randomly. They move up and down quite regularly. They keep in order among themselves, and the varying numbers they produce may result in all sorts of different interpretations by doctors who are unaware of this wide, normal variation.

Since so much of the chemistry and biology of the body changes each day, and changes by as much as tenfold, biologists have gradually come to realize that an animal-whether human or guinea pig-is virtually a different creature, physically and chemically, at different times of day.

This means that we may be more susceptible not only to accidents but also to disease at certain predictable times. It means that a drug taken at one time acts differently than the same drug in the same dose. taken a few hours later or earlier.

Chronobiology has already begun to transform the way biological research is conducted, and it is expected to have a major effect on all of medicine as well. It can alter results at every stage of practice, from preventing time-linked illnesses, to improving diagnoses, to improving treatment. Some of the conventional medical rules of thumb may have to be abandoned; for example, drugs are now given three times a day or four times a day, completely without regard for the differing effects they produce at different hours.

The evidence from the laboratory is clear: When rats are given a nearly lethal dose of amphetamine, their survival depends chiefly on what time the agent is given. At one time in the animal's circadian cycle, six percent of them die. At another time, 78 percent die.

Certain insects have now been found to be far more vulnerable to some commonly used insecticides in the afternoon than at any other times. Rats given a sleep-causing drug will nap for about 50 minutes when the drug is given at one time, but will sleep twice as long when the drug is given later.

High levels of noise usually cause convulsions in animals. But the probability that all animal will be thus afflicted varies according to its body's clock. At the worst time, the probability is 100 percent higher than the daily mean. At the best time, it is 80 percent lower than the daily mean.

In yet another study, rats were given enough phenobarbital to kill at least half of them. But during the most favorable time. none of the animals died. At the least favorable time, the same dose killed all of them. The list could go on. There are already many drugs and active chemicals whose ability to kill or to cure has been shown to swing widely with the rhythms of an animal's body. There are, in short, windows' of daily drug resistance and effectiveness.

Because of this, chronobiologists say, the results of some previous drug and cancer research studies are now dubious. Chronobiologists suggest that studies of toxicity, cspecially of the behavioral effects of toxic agents, must now be completely redone. At the very least, the conduct of scientific research must be changed for all future studies. Time must now be included as a major factor in medical and biological equations.

Colin Pittendrigh, among the most respected of the biological researchers in the field, says that this kind of biological research has begun to have all important impact on medicine. "There are some very important findings, principally from Franz Halberg and his people, on the time-of-day dependence of drug action. That is a firstrate result ... but I have talked to good pharmacologists, and most of them don't yet know the facts. That is worth reporting: The pharmacological fraternity is not informed on what's been found.'

One thing that Pittendrigh suggests should be done immediately is change the federal --sidelines that govern research. The time of an animal's cycle must be taken into account in the research that determines the safety and effectiveness of drugs, he says. Federal agencies provide most of the money for basic research and they also set the guidelines. Thus far, the government has not recognized chronobiology as a major variable in setting guideline policy, and it now appears that a significant factor is being left out of most of the testing.

Despite the evidence that has built up, the common practice in laboratory testing includes such hazardous actions as testing nocturnal animals in the daytime; testing animals that have not had time to adjust to a new laboratory environment; testing animals that are kept in crowded cages, a condition which has been shown to cause aitered internal rhythms among ammals; testing animals in light and dark cycles that are unregulated testing animals in conditions subka to frequent disturbances, such as turning on lights during a dark phase; and testing animals whose feeding schedules are not fixed and recorded. All of these can cause altered rhythms in animals, but none are mentioned in federal research guidelines. Meanwhile, many far less important factors are mentioned.

Lawrence D. Scheving at the University of Arkansas Medical Center,

whose work has established chronobiological effects for a nurnber of drugs, says that time is a variable important enough to affect the validity of some experiments and the accuracy of many.

"We are very careful about controlling the sex. weight. age, and other things. Time is equally important if not more important than the other variables that we rigorously control for,' Scheving says.

One kind of study commonly carried out in toxicology laboratories is the "LD50" study. Animals arc injected with a poisonous agent to determine what dose is lethal to 50 percent of the animals. Commonly, rats for testing may be flown in from halfway across the country, hurriedly brought to the lab, and given the lethal compound. There is no quarantine, no adjustment to environment, no reg@lar feeding n. and no attention to the fight@rk cycle. Similar tests for the same drug often provide results that vary as much as 100 percent. Paying no attention to an animal's cycle, says Morris Cranmer, the former director of the National Center for Toxicological Research, results in studies that are consistently inaccurate. "This means the variation in experiments is increased-and as you increase the variation, you decrease the resolving power of the experiments. Thatis my concern," says Cranmer.

He added another reason to tp' biological time into account, a 1 son that may be even more important than inaccurate results. If a strong effect of biological time is ignored by federal regulations, food and drug manufacturers could use to their own advantage an animal's resistance to toxic effects. Toxicity tests could be designed around an animal's highest tolerance period so that resulting effects would be the most negligible.

The attitude of federal officials varies greatly. William D'Aguanno, the Food and Drug Administration's officer on the toxicology of n ew dr, -igs, says the FDA has no policy on chronobiology. But the attitude of Cranmer's successor at the National Center for Toxicological Research may be seen as a bellwether. Thomas Cairns came into the NCTR when that agency was about to conduct a series of experiments that would have tested the importance of biological time in toxicology. That study got scrapped in the transition from Cranmer's to Cairns' administration. The matter was left there, untouched, for two years. A reporter recently asked Cairns about the importance of chronobiology. He was not up on the literature, he said, and felt that it was probably not very important. But he requested a few days to look into the matter.

Cairns has now begun to worry about the effects chronobiology may have on scientific research funded by the government and on industrial research as well. "My problem is ... that this could easily be used in reverse to a manufacturer's benefit."

A good example of the way in which chronobiology might possibly change medical practice is in the treatment of high blood pressure. Howard Levine, chief of medicine at New Britain General Hospital in Connecticut, and long interested in chronobiology, suffers from high blood pressure. But, he says, "diagnosis of my condition was delayed a couple of years because I used to go to the doctor in the morning. That is when my blood pressure was at its circadian low, and so it seemed normal when actually it wasn't."

Levine also found that late at night the percentage of red blood cells in his blood normally dropped by about four and a half percent, and the total volume of red blood cells in his blood changed by almost ten percent. A five-percent drop,n red blood cells often prom pts a transfusion in a hospital setting.

Frederic Bartter, an eminent endocrinologist at the University of Texas who has done numerous studies of drugs, blood pressure, and their rhythms, points out that every drug that has ever been explored for the rhythmicity of its action has been found to have such rhythms." In normal practice, it Is quite likely that physicians are prescribing drug doses that are anywhere from half to twice as much as a patient needs.

Once a patient's 24-hour rhythm is established, blood pressure need not be measured repeatedly because the pattern is predictable. "Then you can know to what extent a given medication will take away those peaks:' says Bartter.

Chronology of chronobiology

The curiosity about rhythmical events in nature is ancient, but probably the first experimental test of the idea occurred in 1729. The French astronomer jean de Mairan had become curious about a heliotrope plant that opened its leaves to the morning light and closed them at dusk. He discovered that the opening and closing was apparently not controlled by the light in the plant's environment.

Observations like de Mairan's were recorded again and again over the next two centuries by scientists in many fields. At the beginning of this century a major question was whether the cycles In plant behavior were being regulated by a clock within the plant, or by some outside rhythm such as night and day or changing temperature. Experiments that attempted to disrupt the 24-hour rhythms of plants, and later insects and animals, failed. The cycles persisted despite environmental changes.

It was Colin Pittendrigh of Stanford who, in the middle 1950s, finally put into clear terms all the evidence collected up to that time. He and others had proved that clocks' exist in life forms as simple as single-celled animals and as complex as man. Internal clocks, he said, are a fundamental property of life.

While clocks are definitely internal, they may follow external rhythms, be reset by external rhythms, or be disrupted by them. It is, he said, as if two oscillators were operating-one inside the body and one outside.

Most recent research in the field has centered on one main question: Where within the body is the clockwork? Is it an organ or one function of an organ? Is it within the cells, and if so, where within the cells?

The sophisticated clocks found operating in single celled animals settled at least part of the question by proving that one need not look to higher life forms for a fully operating biological timer. Two competing lines of recent research have tried to locate the clock mechanism either in the membrane of the cell, or in the process of protein manufacture.

The first approach holds that the membranes of a cell function by opening and closing channels through which chemicals pass. The process is thought to be electrical: When a certain number of ions build up on one side of a membrane, the flow across it is shut off until the ionic concentration drops again, triggering a feedback mechanism which starts the flow of material through membrane channels again. This theoretical model for an open-and-close rhythm within the cell may be the oscillator, the heart of the ticking clock.

The other approach puts the rate of protein-making at the center of the clockwork. Research has shown again and again that when cells are flooded with substances that foul up the manufacture of proteins, the cells' clocks are reset to a new time. But until recently, it was not certain that the clock was reset only because the protein-making process was disturbed.

Now, in research just completed, Jerry Feldman a former student of Pittendrigh, has demonstrated that one particular chemical that slows the clock does so specifically because it damages protein-making. Cyc!@heximide injected into the ordinary strain of Neurospora fungus normally will foul up the cell's internal clock. But Feldman used a mutant strain of Neurospora, one in which cycloheximide does not damage the protein-making machinery. He wanted to see how the cell's clocks would react to doses of cycloheximide.

Since the protein machinery is unaffected by the chemical, the cell clock should run normally despite the addition of cycloheximide. If the clock changed, however, it would mean that something besides the protein machinery functioned as the clock.

The clocks of the fungus ran normally. 'We proved that protein synthesis is necessary for the clock to run, "he said .'Of course, 'he added 'since proteins are also important to the function of cell membranes, it is probable that both approaches are correct, differing only in emphasis. The one may depend on the other, and thus the two approaches merge."

CAROL EZZELL

After 150 such 'training" cycles, the snail finds itself in a second refrigerated chamber, now with the eye of a video camera staring at it f rom below. This time, when bursts of light flash, an interesting thing happens behind the closed refrigerator door: The animal tenses its foot without being shaken. Ordinarily, light alone would never use that response," says Daniel L Alkon, chief of neural systems at the National Institute of Neurological Disorders and Stroke (NINDS) in Bethesda Md. But it the light appears repeatedly just before and during shaking, this snail, called Hermissenda crassicornis, eventu ally learns to contract its foot when light flashes, "just as Pavlov's dog would salivate when the bell occurred, as if the-smell of meat were there," Alkon says.

For the past 20 years, Alkon and a number of other researchers have been studying the nervous systems of marine snails-and thoseof ratsand rabbits-in a quest for the molecular mechanisms of memory Their searches have led them to a molecule called protein kinase C (PKC) in the surface membranes of nerve cells.

PKC exists in all animal cells, where it plays a role in such diverse physiological processes as growth, blood clotting and the action of hormones. The molecule was first discovered in the early 1970s by a Japanese scientist; in 1979, researchers. found that it acts by tacking a phosphate group onto specific sites on other molecules. The added phosphate changes the function of those molecules, increasing or decreasing their level of activity

One of the first direct clues that PKC might underlie learning and memory came in 1986. Joseph Farley of Princeton (N.J.) University observed that injections of PKC, or of a chemical known to activate PKC, excite light-receptor nerve cells in the eyes of H. crassicornis. This excitation mimics that induced by the light-and-shake regimen: The nerve cells open pores in their membranes that absorb calcium, and close other pores that expel potassium. Using tiny electrodes to track this process, Farley observed that it reversed the normal negative charge inside the cells.

Six years earlier, Alkon had shown that the electrochemical current in neurons changes as an animal learns. He and Joseph Neary, now at the University of Miami, went on to demonstrate that a protein requiring calcium is involved in learning. Because chemicals like PKC mimic the cellular changes of learning, Alkon and Farley launched separate studies investigating PKC as the agent behind those learning-induced current alterations. They proposed that PKC contributes to learning by somehow closing potassium pores, priming the neurons to react more strongly to a new stimulus.

Alkon and Farley reasoned that if a single molecule was responsible for learning and memory, its appearance, disappearance and reappearance should coincide with learning, forgetting and remembering. The molecule might also bring about structural changes in neurons so that they branched to communicate with other neurons in different ways. Moreover, the learning agent would likely prove active in only one region a neuron at a time, so that one neuron would have the capacity to hold multiple memories.

Over the past 18 months, evidence has piledupinsupportofthetheorythatPKC orchestrates neuronal functions necessary for learning and memory 'I have no doubt PKC is central to learning and memory in the models we have looked at," says Alkon, because we've used so many different measures, and have so many different pieces of evidence that are consistent" with PKCs important role.

Farley, now at Indiana University in Bloomington, agrees. The most compelling evidence, he says, comes f rom experiments with marine snails.

"I think it's reasonably clear you can mimic learning [in these snails] using PKC:' Farley says, noting that "inhibitors of PKC will block those changes.' Moreover, his data suggest that "ongoing PKC activation is also necessary for the maintenance of memory in H. crassicornis."

Terry J. Crow, who also works with the colorful marine snail, says the link between PKC and learning is gaining acceptance among other neuroscientists. 'All of the things we have done here suggest that PKC is sufficient to get the neural changes involved in learning going," says Crow, a neurobiologist at the University of Texas Medical School at Houston.

Ayear ago, he reported studies demonstrating that a chemical that inhibits protein synthesis also prevents sea snails from remembering a training cycle for more than one hour. Crow's experiments differed from Alkon's because these snails received only one flash of light, immediately followed by an injection of serotonin-a neurotransmitterthatcrow believes is also crucial to memory

Alkon interprets the findings difterentty. Even though Crow and

AFarley have evidence that the neurons feeding signals to the light receptors in the eyes of sea snails contain serotonin, Alkon remains unconvinced that serotonin plays a primary role in learning. He has never found serotonin in cells adjacent to sea-snail light receptors, and he contends that the serotonin detected by Crow and Farley must arise elsewhere in the brain.

Alkon suspects that a different neurotransmitter - called GABA, for gammaaminobutyric acid - provides half of the one-two punch that causes H. crassicornis to link two events such as light and shaking.

Neurophysiologists Juan V SanchezAndres and Ren6 Etcheberrigaray, working in Alkon's laboratory, have used two different techniques to measure the effects of GABA on light-receptor cells taken from H. crassicomis eyes. They found that GABA causes the receptor cells to close their potassium pores, exciting them in a manner that mimics the effects of learning.

Other researchers in Alkorfs lab have detected GABA in the tiny, hair-bearing cells of statocysts - organs used by H. crassicorn,'-- to sense motions such as shaking. In addition, says Alkon, they have demonstrated that chemicals that inhibit PKC also block the memory-mimicking effects of GABA.

'A lotof people may be excited by this,. says Alkon. In previous studies, he says, GABA had been shown to inhibit neurons, not to excite them. He and senior coauthor Louis Matzel will present the

On the basis of his group's GABA findings, Alkon has constructed a theoretical model of the sequence of events occurring in the outer membranes of a snail's light-receptor nerve cells when the animal learns to connect light flashes with being shaken. According to his model, an incoming light flash triggers a series of impulses in the neuron, accompanied by an influx of calcium. Then the shaking causes the snail's statocyst to release GABA, which binds to the neuron. Both events cause PKC stored inside the cell to move to the cell membrane, where the PKC shuts down the potassium channels by acting through one member of a class of membrane agents called G proteins. When the potassium channels close, the neuron becomes more excitable, so that light later produces the same behavioral response as shaking.

Farley challenges Alkorfs assertion that a G protein is necessary to help PK( close potassium channels and keep z neuron excited for learning. -in oul model, PKC acts on the channels di rectly ... There's no need for a G protein,' Farley says-

Crow, Farley and Alkon do agree, how ever, that PKC is responsible for th( increased excitability of neurons tha have learned.'The PKC-induced change: in learning last for many days, and some times even for weeks," notes Alkon.

But how do the relatively short-tern effects of PKC get translated into long term memories?

More recently, Nelson has uncovered evidence that the G protein cited by Alkon exerts control over mRNA. 1t turns out that this G protein regulates the turnover of mRNA or the readout of mRNA,Alkon says.'That's very exciting because it suggests the G protein not only has effects on the (potassium] channels, but also affects the synthesis proteins. Nelson and Alkon are now preparing to publish the finding.

What does protein synthesi have to do with learning? Alkon thinks long-term memory depends upon "hard-wiring changes that strengthen some connections between neurons while reducing others. These structural changes occur in tne dendrites, the branching finger through which neurons receive incoming electrical impulses. To change their branching pattern. neurons must manufacture new proteins.

In Alkon's senario, learning activates neurons PKC, which in turn activates a protein. The G protein then closes the potassium channels, keeping the neuron

excited for short term memory. The protein also regulates protein synthesis which 'hard-wires" the memory over the long term by changing the neurons branching structure.

Farley questions whether changes in neuron branching build long-term memories. "I'm a little skeptical about these changes in cell volume that are purported to be happening [as a result of learning],' he says.

Experiments at Alkon's laboratory back up this scenario, however. In the February L990 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (Vol-87, No.4), Alkon and several co-workers report the results of injecting dye into lightreceptor cells of trained and untrained snails. The dye revealed that the neurons of snails trained to associate light and shaking had fewer and more condensed branches than did those of untrained snails, suggesting that learning could reroute a nerve cell's branching.

Such rerouting should be reflected in fluctuations in the shipment of new cell membrane from the body of the cell to the branch tips, Alkon reasoned. Last year, Simon Moshiach from Alkotn's lab, together with Nelson and Sanchez-Andres, demonstrated just such a change. With one squirt of G protein extracted from a snail, they slowed the flow of new protein globules along the axon-the long "arm" that transmits outgoing messages - of a large nerve cell taken from a crab.

Although Alkon contends that long term memory probably requires changes in neuron structure, he also finds evidence that PKC is involved. Working with Matzel, now at Rutgers University in New Brunswick, N.J., he turned up evidence that PKCs effects can persist for weeks after H. crassicomis has learned.

Matzel trained the snails with just enough paired cycles of light and shaking that they learned to associate the two stimuli. He did not repeat the cycles over and over to reinforce the animals' memories, however. After about one week, the snails forgot the association: Light alone no longer caused their feet to tense. But when Matzel retained the animals.after waiting two weeks, he found that they could relearn the association after just a handful of trials.

'Even though they had forgotten, they retained some memory, because they relearned much more quickly," Alkon says-

With increasing evidence linking learning and memory in Alkon's team began looking for a similar link in higher animals.

In 1988, Alkotn's co-worker Barry Bank, with colleagues from NINDS and Yale University, used a radioactive stain to trace the activation of PKC in rabbits. They found that rabbits trained to associateaparticulartonewithamildelectrical shock near their eyes eventually learned todropa protective membraneovertheir eyes whenever they heard the tone. The stain revealed that the trained rabbits harbored increased levels of PKC in the hippocampus of the brain. Previous st6d-ies involving animals and humans with hippocampal injuries had shown that this area of the brain is crucial to maintaining memory for many days.

James L. Olds of NINDS went one step farther by precisely tracing the movement of PKC throughout nerve cells in the rabbit hippocampus. He found that PKC levels increased in the cell bodies of the neurons one day after the rabbits had undergone training, and that the PKC moved to the dendrite membranes three days after training. These results support the finding in sea snails that after learning, PKC moves from the body of a nerve cell and into its membrane.

Working with David Olton of Johns Hopkins University in Baltimore, Olds has also trained rats swimming through a tank of water to distinguish unstable platforms from those stable enough to allow the animals to climb out and dry off. Rats given cues about which platforms were stable had lower hippocampal levels of PKC than did rats that learned the difference through trial and error, the researchers report In the November 1990 Jou OF NEuRosa

Recently Olds and Sanchez-Andres have begun to investigate the PKC changes that occur early in an animal's development. By staining brain slices from baby rabbits not yet old enough to haveopened theireyes. they have discovered that most of the PKC in the hippocampal neurons resides in two tracks flanking the cell body in contrast. the hippocampal neurons of older rabbits have a single, diffuse track of PKC situated around the dendrites. When the baby rabbits open their eyes and begin exploring their environment at about 10 days of age. their PKC moves into the dendrites, perhaps programming their memories, Olds says.

He speculates that very young rabbits@ hoard PKC near the cell bodies because they haverft yet had a chance to learn anything about their surroundings. Then, when they open their eyes, their brains are flooded with new information worthremembering-achangethatkicks the PKC memory pathway into action.

Olds notes that many other researchers have now implicated PKC in the development of neurons. "I'm trying to figure out the role of PKC in learning and postnatal development,' he says. "I'm convinced that similar molecular mechanisms are involved.'

Others are investigating PKCs involvement in human disorders, such as Alzheimer's disease, that erode both memory and learning capacity. A group led by Tsunao Saitoh at the University of California, San Diego, reports in the July 1990 JOURNAL OF NEUROSCIENCE that the brains of 11 deceased Alzheimer's victims contained only half as much PKC as the brains of seven people who had died of other causes. The results confirm previous findings by SaitoWs team (based on a less specific method for measuring PKC), which had hinted that PKC levels drop in Alzheimer's patients.

Saitoh and his colleagues ruled out the possibility that the reductions in PKC resulted from the overall death of neurons in Alzheimer's patients by measuring the PKC levels of connective-tissue cells in the autopsied brains. These cells also showed lower-than-expected amounts of PKC, they found.

Researchers who are studying the humble sea snail find encouragement in thediscoverythat PKC is linked to human learning and memory It proves they aren't chasing down blind alleys in their for the molecular Holy Grail of memory At the same time, they're cau@ about attributing the entire orchestration of human learning to PKC.

"We don't know about other molecules, such as G proteins, that are likely to be involved In learning [in humans and other higher animals],' says Alkon. "Molecular pathways are very complex, and there are undoubtedly hosts of other actors in this drama that we haven't yet met

Adds Farley, 'There's little doubt that PKC is Involved in an important way, but It's certainly not the entire story!'

Differences Found In the Brain Anatomy of Men and Women

Daniel Goleman

Researchers who study the brain have discovered that it differs anatomically in men and women in ways that may underlie differences in mental abilities.

The findings, although based on small-scale studies and still very preliminary, are potentially of great significance. If there are subtle differences in anatomical structure between men's and wonien's brains, it would help explain why women recover more quickly and more often from certain kinds of brain damage than do men, and perhaos help guide treatment.

The findings could also aid scientists in understanding why more boys than girls have problems like dyslexia, and why women on average have superior verbal abilities to men. Researchers have not yet found anything to explain the tendency of men to do better on tasks involving spatial relationships.

The new findings are emerging from the growing field of the neuropsychology of sex differences. Specialists in the discipline met at the New York Academy of Sciences last month to present their latest data.

Research on sex differences in the brain has been a controversial topic, almost taboo for a time. Some feminists fear that any differences in brain structure found might be used against women by those who would cite the difference to explain "deficiencies" that are actually due to social bias. And some researchers argue that differences in the brain are simply due to environmental influences, such as girls being discouraged from taking math seriously.

The new research is producing a complex picture of the brain in which differences in anatomical structure seem to lead to advantages in performance on certain mental tasks. The researchers emphasize, however, that it is not at all clear that education or experience do not override what differences in brain structure contribute to the normal variation in abilities. Moreover, they note that the brains of men and women are far more similar than different.

Still, in the most significant new findings, researchers are reporting that parts of the corpus callosum, the fibers that connect the left and right hemispheres of the brain, are larger in women than men. The finding is surprising because, over all, male brains-including the corpus callosum as a whole-are larger than those of females, presumably because men tend to be bigger on average than women.

Because the corpus callosum ties togethcr so many parts of the brain, a difference there suggests far more widespread disparities between men and women in the anatomical structure of other parts of the brain.

"This anato ndcal difference is probably just the tip of the iceberg," said Sandra Witelson, a neuropsychologist at McMaster University medical school in Hamilton, Ontario, who did the study. "it probably reflects differewes in many parts of the brain which we have not yet even gotten a glimpse of. The anat(xny of nien's and women's brains may be far more different than we suspect."

'Me part of the brain which Dr. Witelson discovered is larger in women is in the isthmus, a narrow part of the callosum toward the back. Her findings, reported in March at the New York Academy of Sciences meeting, will be published in the journal Brain.

Dr. Witelson's findings on the isthmus are based on studies of 50 brains, 15 male and 35 female. The brains examined were of patients who had been given routine neuropsychological tests before they died.

"Witelson's findings are potentially quite important, but it's not clear what they mean," said Bruce McEwen, a neuroscientist at Rockefeller University. "In the brain, bigger doesn't always mean better."

In 1982 a different area of the corpus callosum, the splenium, was reported by researchers to be larger in women than in men. But that study was based on only 14 brains, five of which were female. Since then, some researchers, including Dr. Witelson, have failed to find the reported difference, while others have.

Since such differences in brain structure can be subtle and vary greatly from person to person, it can take the close examination of hundreds of brains before neuroanatornists are convinced. But other neuroscientists say the findings are convincing enough to encourage them to do tests of their own.

Both the splenium and the isthmus are located toward the rear of the corpus cauosum. This part of the corpus callosum ties together the cortical areas on each side of the brain that control some aspects of speech, such as the comprehension of spoken lan-

guage, and the perception of spatial relationships

From The New York Times, April ii. IM. pp. Cl. C6. 1991 by The Now York Times Company. Reorinted by

"The isthmus connects the verbal and spatial centers on the right and left hemispheres, sending information both ways-it's a two-way highway," Dr. Witelson said. The larger isthmus in women is thought to be related to women's superiority on some tests of verbal intelligence. It is unclear what. if anything the isthmus might have to do with the advantage of men on tests of spatial relations.

The small differences in abilities between the sexes have long puzzled researchers.

On examinations like the Scholastic Aptitude Test, which measures overall verbal and mathematical abilities, sex diffe in scores have been declining. But for certain specific abilities, the sex differences are still notable, rers say.

While these differences are still the subject of intense controversy, most researchers agree that women generally show advantages over men in certain verbal abilities. For instance, on average, girls begin to speak earlier than boys and women are more fluent with words than men, and make fewer mistakes in grammar and pronunciation.

On the other hand, men, on average, tend to be better than women o certain spatial tasks, such as drawing maps of places they have been and rotating imagined geometric images in their minds' eye-a skill useful in mathematics, engineering and architecture.

Of course, the advantages of each sex are only on average. There are individual men who do as well as the best women on verbal tests, and women who do as well as the best men on spatial tasks.

One of the first studies that directly links the relatively larger parts of women's corpus callosw-ns to superior verbal abilities was reported at the meeting of the New York Academy of Sciences by Melissa Hines, a neuropsychologist at the University of California at Los Angeles medical school.

Dr. Hines and her associates used magnetic resonance imaging, a method that uses electrical fields generated by

the brain, to measure the brain anatomy of 29 women. They found that the larger the splenium in the women, the better they were on tests of verbal fluency.

There was no relationship, however, between the size of their splenium and their scores on tests of spatial abilities, suggesting that differences in those abilities are related to anatomical structures in some other part of the brain or have nothing to do with anatomy "The size of the splenium," Dr. Hines said, "may provide an anatomical basis for increased communication between the hemispheres, and perhaps as a consequence, increased language abilities."

Researchers now speculate that the larger portions of the corpus callosum in women may allow for stronger connections between the parts of women's brains that are involved in speech than is true for men.

"Although we are not sure what a bigger overall isthmus means in terms of microscopic brain structure, it does suggest greater interhemispheric communication in women," Dr. Witelson said. "But if it does have something to do with the cognitive differences between the sexes, it will certainly turn out to be a complex story."

Part of that complexity has to do with explaining why, despite the bigger isthmus, women tend to do less well than men in spatial abilities, even though the isthmus connects the brain's spatial centers, too.

"Bigger isn't necessarily better, but it certainly means that it's different," Dr. Witelson said.

A variety of other differences in the brain have been detected by the researchers in their recent studies.

"How our brains do the same thing, namely use the right hand, may differ between the sexes," Dr. Witelson said.

She also found that the overall size of the callosum, particularly the front

part, decreases in size between 40 and 70 years of age in men, but remains the same in women.

Several converging lines of evidence from other studies suggest th ' at the brain centers for language are more centralized in men than in women.

One study involved cerebral blood flow, which was measured while men and women listened to words that earphones directed to one ear or the other. The research, conducted by Cecile Naylor, a neuropsychologist at Bownian Gray School of Medicine in Winston-Salem, N.C., showed that the speech centers in women's brains were connected to more areas both within and between each hemisphere.

This puts men at a relative disadvantage in recovering from certain kinds of brain damage, such as strokes, when they cause lesions in the speech centers on the left side of the brain Women with similar lesions, by contrast, are better able to recover speech abilities, perhaps because stronger connections between the hemispheres allow them to compensate more readily for damage on the left side of the brain by relying on similar speech centers on the right.

In the current issue of the Journal of Neuroscienoe, Roger Gorski, a neuroscientist at UC.L.A., reported finding that parts of the hypothalamus are significantly bigger in male rats than in female ones, even though the size of the overall brain is the same in sexes.

And Dr. McEwen, working with colleagues at Rockefeller University, has found a sex difference in the structure of neurons in part of the hippocampus that relays messages from areas of cortex.

Dr. McEwen, working with rats' brains, found that females have more branches on their dendfites, which receive chemical messages from other neurons, than do males. Males, on the other hand, have more spines on their dendrites, which also receive messages from other neurons. These differences in structure may mean differing patterns of electrical activity during brain function, he said.

"We were surprised to find any difference at all, and, ftmkly, don't understand the implications for differences in brain function," Dr. McEwen said. "But we'd expect to find the same differences in huams; across the board, findings in rodents have had corollaries in the human brain."

Matthew Allen Gonda

In 1984, a previously unknown virus was isolated from human blood. Named HTLV-111 (human T-cell lymphotropic III), the virus selectively a specific group of white blood cells crucial to the body's immune response. Soon generally recognized as the causative agent of acquired immunodeficiency syndrome, or AlDS, the virus was later discovered to have an affinity for infecting cells of the brain as well.

Although the AIDS disease process has proved to be enormously complex and often baffling-there is no complete parallel for it among the other viral diseases of humans-we have learned a great deal about the virus's molecular biology and structure in a very short time. Structurally and biochemically, HTLV-III belongs to the retrovirus family, a unique subgroup of viruses found not only in humans but also in many animals, from reptiles to primates. Like other viruses, retroviruses don't always cause disease in their hosts.

Also like other viruses, retroviruses are not really living organisms. Lacking the machinery and the energy-generating capabilities to manufacture progeny, they are perhaps best described as infectious chemicals made up of a sticky protein coat encapsulating a genome (the DNA or RNA blueprint for constructing more viruses). Incapable of growth and division on their own, viruses exploit the cells of living organisms to perform these functions for them. Infection occurs when, via highly specific receptors on its protein coat, a virus attaches itself to and penetrates a susceptible cell. Once inside, it is read and reproduced by the host's manufacturing machinery. Sometimes the cell is killed during virus replication; but before its demise, it has released a new generation of viruses into the host's system.

Retroviruses have evolved a particularly effective variation on this parasitic theme. Unusual because their genomes are composed of RNA (in most living things, including most viruses, genome! are composed of DNA), retroviruses also possess a gene for a unique enzyme, reverse transcriptase. When the retrovirus attaches itself to and penetrates a reverse transcriptase transcribes retrovirus's genetic information from RNA into DNA. The host, often perceiving this new DNA to be its own genetic material, integrates it into its own chromosomes. Once in this new habitat, the retrovirus may be reproduced or it may remain dormant for weeks, months or even years. The virus stays in the chromosomes for the life of the cell, that is, until the cell has been killed by the infection eliminated by the immune system, or removed after senescence. The association is permanent; every time host cells reproduce, they also reproduce retrovirus DNA, even in the absence of new virus.

Some mouse and chicken retroviruses have assured themselves of even longer relationships with their hosts. Because in a past event they infected and were integrated into the host's germ cells (that is sperm and egg or their precursor cells) they are now automatically transmitted to, the next generation of host animals with out an infectious cycle. There are no known methods of eliminating these so called endogenous viruses.

Other retroviruses are exogenous-that is, acquired from the outside. The AIDS virus, passed from person to person (or, from pregnant woman to fetus) via infected blood or body fluids, is of this type Exogenous or endogenous, however, retro virus infections have one feature in common; infected individuals remain infected (though not necessarily ill) for life.

Before the discovery of the AlDS virus only two other retroviruses had ever been isolated in human beings. These-the human T-cell leukemia viruses, HTLV-1 and 11-belong to the oncovirus subfamily of retroviruses, so called because they are oncogenic (tumor producing in their host). Like the AIDS virus, they attack T-4 lymphocytes, the white blood cells that begin the immune reaction. The question therefore arose early as to whether the AIDS virus was also an oncovirus. At first, th idea seemed plausible, because of the properties shared with the leukemia viruses, the most prominent of which was their affinity for T-cells. In addition, the AIDS virus was suspected of causing Kaposi's sarcoma, a rare cancer of the skin's blood vessels, from which many AIDS victims suffer. Further investigation, however, made it clear that HTLVIII did not directly cause Kaposi's sarcoma. Rather, the tumors were arising opportunistically because of the underlying immune deficiency, just as they do in organ-transplant patients who are given immunosuppressive drugs.

If the AlDS virus was not an oncovirus, what was it? Investigators began to look for similarities in the two other known rctrovirus subfanillies-the lentiviruses and the spumiviruses. (There was also the possibility that it belonged to a new group of retroviruses not previously identified.) The spumiviruses, or foamy viruses although they had not been thoroughly studied, were ruled out quickly; they were not known to cause disease, and structurally, they differed sharply from both the leukemia viruses and from the AIDS virus.

Important clues to the identity of the virus were already apparent, however. Most important was that the AIDS virus did not cause cancerous proliferations but instead brought about cell-killing (cytolytic) events. This cytolytic propensity is one of several distinguishing properties of the lentiviruses. Called "slow" viruses for their slow but persistent rate of replication, the lentiviruscs eventually induce debilitating diseases, although years may pass between the initial infection and the onset of symptoms. Since the AIDS virus also is associated with the slow evolution of a lethal debilitating disease, this was a second family resemblance. Firmer evidence came from electron microscope pictures; HTLV-III strikingly resembled the visna virus, a lentivirus that infects sheep.

Lentiviruses that have since been identified in other animals induce a variety of disease syndromes. Caprine (goat) arthritis encephalitis virus, which is genetically very closely related to visna, causes crippling arthritis, paralysis, and encephalitis in goats. Horses are vulnerable to a lentiviral agent called equine infectious anemia virus, which causes intermittent anemia, bouts of fever, and immune-complex glomerulonephritis, an inflammatory disease of the kidneys occurring secondary to the infection. The lentivirus of cows, bovine visnalike virus, also affects the lymph system and causes persistent lymphocytosis, an excessive production of white blood cells.

When observed under an electron microscope, all of the lentiviruses, including the AIDS virus, share a common physical structure. Each infects cells of the immune system, although the specific target cell and the level of interference with the host's immune response differ from species to species. Visna and Caprine arthritis encephalitis viruses seem to attack the large white blood cells, the monocytes and macrophages. These cells normally devour foreign bacteria and cellular debris and are a first line of defense against infection. Besides attacking the T-cells, HTLVIII also infects monocytes and macrophages, as well as antibody-producing lymphocytes. Whether other cells of goats and sheep are affected by lentiviruses is not known, since their immune systems have not been as intensively studied as that of humans.

Further analysis of the relationship between the AIDS virus and visna virus awaited direct comparison of their genetic sequences. For if it could be proved that the AIDS virus is genetically related to the lentiviruses, some of the disease's mysterious processes would begin to make sense. DNA hybridization using cloned DNAs of the viruses, an effective way of grossly estimating genetic relatedness, revealed that the AIDS virus and the lentivirus resemble one another even on the very basic level of their DNA sequences. Of the several genetic likenesses investigators saw, the most dramatic was the similarity in the gene for coding reverse transcriptase. This gene, in fact, has changed the least in the evolution of retroviruses, and virologists now depend upon it to determine phylogenetic formation for the group. Overall, the AIDS virus and visna virus had significantly more DNA sequences in common than either did with any oncovirus tested, including HTLV-1 and HTLV-11.

By this time it was evident that HTLVIII and visna virus were close cousins. But the question of whether HTLV-111 was also related to other lentiviruses awaited testing of other representative species. Equine infectious anemia and caprine arthritis encephalitis virus were subsequently cloned and showed an equal amount of likeness with HTLV-111. Clearly, the AIDS virus was a lentivirus.

Final confirmation came from DNA sequencing, which allows nucleotide-bynucleotide comparisons of the reverse transcriptase gene and the rest of the virus genome. (Each nucleotide represents a single letter of the genetic code.) It demonstrated that the genomes for HTLV-I II and lentiviruses were similar in organization and coded for similar sets of genes in the same order and location. This information was important, because. sequencing determines how the virus is assembled. how it works, and what it looks like.

In 1985, not long after the structural and genetic studies were reported, another

important clinical manifestation of AIDS was recognized. Physicians began to realize that neurological signs and symptoms that they had been seeing in AIDS patients-chronic meningitis, dementia, encephalopathy, loss of motor coordination, and paralysis-were caused directly by the AIDS-virus infection. The findings suggested that HTLV-III was attracted to brain cells as well as to white blood cells. In retrospect, in view of the virus's demonstrated close association with the visna and caprine retroviruses-both of which cause neurological disease-the findings should not have been that surprising.

A great deal was being discovered about HTLV-111, but much of it boded ill for the development of a vaccine. On the one hand, since the virus was exogenous (transmitted from outside), there was an inherent "weakness" in its replicative cycle that could be exploited. Uninfected persons could theoretically be protected via vaccination, as has been done with other horizontally transmitted viral diseases, such as measles, mumps, and smallpox. But the AIDS virus is a retrovirus, and to date an effective vaccine has been made for only one retrovirus, feline Ieukcmia virus, a cancer-causing retrovirus of cats. Although this vaccine has not totally contained the disease-probably because some apparently healthy cats had already been infected-its existence at least raises the possibility that a successful human retrovirus vaccine can also be developed.

Normally, the host immune system counters infection by making protective antibodies that are specially adapted to adhere to and destroy a specific attacking virus. Lentiviruses, however, have developed novel strategies to avoid elimination by the host. Visna virus and equine infecmia virus, for example, undergo rapid changes in the gene responsible for their charactenstic protein coat. This capacity for change, called antigenic drift produces variants of the virus that are not recognized by the host's protective antibodies, which were effective in neutralizing the original strain. The va viruses thus escape destruction and can continue to infect and, sooner or later induce a new cycle of disease. (An analgous process takes place in the envelope of the influenza virus and has created a major stumbling block in obtaining a single ceffective flu vaccine.)

Caprine arthritis encephalitis virus has another means of evading destruction. It evokes a very weak immune response; the antibodies that do respond seem to do so only halfheartedly and do not kill the virus. The AlDS virus seems to act similarly in this respect, and even though antibodies are present, they do not appear to prevent severe disease or predict survival for the patient. Additionally, the envelope gene HTLV-III is quite variable, indicating that both of the mechanisms described may be at work.

Effective vaccines have not been made for any lentiviruses, so that producing an AIDS vaccine is no trivial task. Any knowledge gained about lentivirus disease in animals will contribute to the effective control of AIDS.

HTLV-III was presumably introduced he United States in the 1970s, and was first recognized clinically in 1981. Although no one knows whether the HTLV-III-induced syndrome is a new disease or where it came from, scrologic data are now accumulating to suggest that the virus was in Africa at least a decade before it came to the United States, probably via Haiti. What we don't know is whether the virus was present in humans before the first documented evidence or whether it came from an animal reservoir.

We can only speculate on these possibilities. If the virus was widely present in humans before that time, it must have gone through a genetic change that made it more pathogenic. But there are no data at present to substantiate the coexistence of pathogenic and nonpathogenic forms of the virus. It is hard to believe that the nonpathogenic version could have died out in the few years since giving rise to a pathogenic form.

Another possibility is that there exists a lentivirus family group in animals that resembles the AIDS virus even more losely than already identified lentiviruses and that a virus in this group gave rise to a human variant. A newly isolated virus, called STLV-III (simian T-lymphotropic virus type III), causes an AIDS-like syndrome in the macaque monkey. The simian virus resembles the AIDS virus in growth characteristics and structure, and it is attracted to similar cells. These data suggest that STLV-111 may also be a lentivirus. Moreover, the presence of strongly cross-reactive antibodies to STLV-III in the blood of apparently healthy wild African green monkeys suggests that the virus is not disease producing in one species and quite pathogenic when it is transmitted to another in this case the macaque.

How close is the relationship between the simian virus and the AIDS virus? There is at least the possibility of a monkey retrovirus giving rise to a human variant. Human leukemia virus type I , for instance, has a correlate in a simian virus (Simian leukemia virus type 1), to which it is remarkably similar in terms of DNA. There have been no direct comparisons of the AlDS-virus DNA and its simian counterpart, but serological analyses have provided some evidence that the AlDS virus may be closer to STLV-111 than it is to other lentiviruses. However, the two are not nearly so closely related as the human leukemia virus type I and the simian leukemia virus type 1. Even if STLV-111 crossed from monkeys to humans, it is unlikely to have diverged so much in such a short time, to become HTLV-111.

A better analogy may be found in the sudden appearance of visna virus in sheep, first described by Bjorn Sigurdsson a physician. Before 1933, visna was unknown in Iceland. That year, the government purchased twenty karakul sheep

from a farm near Halle, Germany, where a visnalike virus was endemic. (The disease in Germany was less severe than what was later seen in Iceland.) When the sheep arrived in Iceland, they were put into quarantine for several weeks and then distributed to farms scattered all over the country. At least two of the introduced breed apparently carried the infection at the time of quarantine because by 1935, there were outbreaks in two widely scparated districts. Until 1939, however, no one realized that the disease was an entirely new entity. The losses were enormous. Between 1939 and 1952 at least 150,000 animals died of the infection.

Between 1949 and 1951 all the sheep in the southern part of the island were destroyed in an attempt to control the virus, and ultimately the disease was brought under control. It is now known that visna can be easily transmitted from ewe to kid during feeding, especially through the virus-laden immune cells found in the colostrum, the fluid secreted by the mammary glands before milk appears. Chance abrasions of the skin or mucous membranes are other possible modes of entry, as are the bites of blood-sucking insects, which are implicated in the spread of equine infectious anemia. It could also be that centuries of isolation made Icelandic sheep particularly susceptible to visna.

If HTLV-111 is not a new virus, has not recently jumped into the human species from an animal reservoir, and is not a mutation of a known nonpathogenic virus, what plausible explanation can account for its sudden appearance in humans' Drawing on the parallels with visna, we can make some educated guesses. It is possible that the virus has existed in humans in central Africa for hundreds of thousands of years, but that it resided in an isolated population. Such isolated groups may have coadapted with the virus, lessening the severity of the infection and allowing for mutual coexistence. The persistence of HTLV-111 in this scenario may lie in old customs such as scarification and the sharing of needles used for body marking. Parallels exist in the spread of kuru, a slow-acting and fatal viral disease of the nervous system. Kuru is found only in a specific tribe in New Guinea and is spread exclusively by rites associated with cannibalism.

Demographic factors, too, may have a bearing on the spread of AIDS. In the past thirty years, Africa's tribal and geographical boundaries have broken down as individuals moved toward cities for a variety of reasons. Such changes could have brought an infectious agent into contact with previously unexposed populations both international and local, and the devastating effects of the virus would have been felt more readily, as when visna virus was introduced to Iceland.

Such a pattern of sudden virulence has often been seen when other pathogens have reached unexposed peoples. Exampies include the fatal measles epidemic in the Faroe Islands (1781) and the Fiji Islands (1875), and the devastating effects of smallpox on American Indian populations after contact with Europeans in the sixteenth century. Anthropological studles in central Africa may provide further insights into such a scenario.

Fortunately, the AIDS virus, unlike smallpox or measles, is not easily transmitted, and unlike some other retroviruses, it cannot be transmitted from generation to generation. Therefore, even in the absence of a vaccine, we can expect that preventive measures already in place can effectively prevent the spread of the disease.

With permission from NATURAL HISTORY, Vol. 95 No. 5, May 1986, pp. 78-81. Copyright the American Museum of NaturaI History.

Could other infections or genes boost a symptomless AIDS infection into full-blown AIDS?

JOANNE SILBERNER

ne of the great mysteries of AIDS is why some people who have been 0 infected by the AIDS virus go years - if not their lifetimes-without developing the syndrome. Many AIDS researchers believe one or more additional elements, or cofactors. are necessary to turn an AIDS-virus infection into actual I disease.

According to the U.S. Public Health Service, about I million to 1.5 million people in t@e United States are infected by human immunodeficiency virus (HIV), and roughly 20 to 30 percent of them will develop AIDS within five years. Who among the infected individuals will ij get the syndrome and when that will happen are open questions. Finding a cofactor would enable physicians to identify these people and possibly show how to prevent the progression from infection to illness.

the many possible cofactors been proposed, two of the t candidates are the presence of genetically determined proteins.

Infected individuals, and exposure viruses. If the virus co-infection hypothesis, whose proponents include researchers f rom the National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Md., is true, avoidance of a second virus could be the key to health. But a genetic predisposition, as suggested by researchers at the University of California at San Francisco, would be more difficult to counter.

With most viruses, infection does not always mean a person becomes sick -for example, the majority of people infected with hepatitis B virus or with poliovirus dorft develop symptoms. But while cofactors are evidently an element in the sand other serious viral infections, there has not been a lot of research into the issue, says epidemiologist Harold Jaffe of the Centers for Disease Control in Atlanta. Questions about cofactors "could be asked for lots of other diseases," he says. The sudden, mysterious and deadly onset of the AIDS epidemic has lent the question 'a sense of urgency," he says.

Because many members of the two highest-risk groups, male homosexuals and intravenous drug abusers, have histories of frequent sexually transmitted or blood-borne diseases, some researchers have been investigating whether a second infection can somehow "awaken" the AIDS virus. Recent results from Malcolm Martin and his co-workers at the NIAID provide biological support for the possibility

Martin, Howard E. Gendelman and their co-workers studied the interaction of HIV and other viral infections in cells growing in culture. To avoid the hazards of working with the entire AIDS virus, they used only a segment of HIV's genetic material, linked to a bacterial gene that directs the construction of an easy-totest-for enzyme.

Martin and his colleagues introduced the combination genes into a cell line and followed its activity by monitoring the marker enzyme. When they added any one of several viruses that commonly infect people, they found more of the marker enzyme, indicating that the AIDS virus material was much more active. Martin says subsequent experiments using the entire AIDS virus have confirmed the initial results, which were published in the December Proceedings of the National Academy of Science

(Vol.83, No-24).

The viruses used in the experiment are so different from one another that they could possibly all be acting in the same way, he says. Rather than all the viruses producing an identical protein that travels to the AIDS virus and causes it to reproduce, Martin suggests the non-HIV viruses somehow induce the cell itself to stimulate HIV, perhaps by making the cell produce an HIV-stimulating protein.

Several laboratories, including Martin's, are searching for such a protein. Unfortunately, if the infected cell's own protein is responsible, interceding in the process may be difficult. 'They [the proteins] are probably there for some important normal function," says Martin. Interrupting that function to keep the proteins from stimulating the AIDS virus could cause other problems. 'The more we know,' he says, "the less we know.'

On the other hand, the cell may also be capable of producing other proteins that inhibit the system, Martin suggests. If so, stimulating those proteins could keep HIV quiet. And whatever the mechanism of action of other viral infections, if they are what's kicking off HIV, avoiding them would be a way to avoid getting AIDS.

While Martin's theory holds that a second infection kicks off AIDS, John Ziegler and Daniel P Stites of the University of California at San Francisco suggest that the cofactor is a genetic one. They base their theory on the paucity of active AIDS virus found in full-blown, or 'frank," disease.

"It's very difficult to find infected lymphocytes [white blood cells] in infected blood ,Ziegier says. In frank AIDS. or in 10,000 to I in IOOPW lymphocytes s evidence of viral infection.

The AIDS virus attacks and infects the CD4 cell, a type of white cell, at the location where the CD4 normally' docks' with other cells in the immune system. This docking process is a necessary step in a cascade of events that results in the recognition and neutralization of foreign substances.

In order to attach to the CD4 dock, Zeigler and Stites suggest, the virus must in some way "look" like the second set of cells. And this similarity results in the virus affecting the immune system not only by destroying the cell it infects but also by generating antibodies that attack the immune system in two separate ways.

First, antibodies to the virus also attack the cells that normally link up with the CD4 cells, since the virus and the second set of cells have something in common. According to the hypothesis, these antibodies block the interaction of the CD4s and the other cells - even though neither may be infected by the virus. Second, the virus-prompted antibody also triggers the production of other antibodies against both itself and the CD4s, again including those that have not been infected by the AIDS virus. As a result, an entire and vital arm of the immune system is wiped out.

In this way,' says Ziegler, "just a handful of HIV could kick off immune system self-destruction."

Genetics comes into play because the proteins on the immune system cells to which the CD4s attach differ from person to person, and these proteins are inherited. People whose proteins "look' like proteins on the surface of HIV would develop the two sets of antibodies that attack the immune system, and go from infection to full-blown AIDS. People whose proteins differ markedly from the HIV strain would be spared.

If the hypothesis is proven true, it has both positive and negative implications for therapy. The immune self-attack aspect suggests that toning down the immune response could help. Therapeutically, 'you'd want to think of ways to remove antibodies to see what happens to patients,' says Ziegler. French researchers already have tried damping the immune response with cyclosporine, and a small LLS trial with cyclosporine began recently

But it would also throw a wrench into vaccine development. If the part of that is similar to the antigen-presenting cells were used as a vaccine, the antibodies generated against the vaccine material would also be capable of attacking the antigen-presenting cells themselves. Such a vaccine would have the unfortunate result of destroying a normal, necessary arm of the immune system.

Two discoveries would help prove the genetic hypothesis: identifying a single antibody that attacks both HIV and the cells to which the CD4s attach, and the preponderance in AIDS patients of particular classes of proteins on white cells that differ from those in people who are infected but have not developed the syndrome. Collaborators of Ziegier's at UCSF are no win the process of looking for similar classes of proteins among people with AIDS, and there have already been several reports from other laboratories indicating that such clustering exists. Ziegle'r's collaborators and other U.S. laboratories are also checking an antibody against white cells found in people with AIDS to see if it attacks HIV as well.

Ziegler. 'Obviously everything isn't going to be explained by genetics. But if it lies there we should be able to find it'

Ziegier's and Martin's theories' aren't mutually exclusive - they couId each be at work in different people. Now are genetics and viral infections the only candidates that have been suggested. Ziegler in fact, has worked with UCSF's Jay Levy on a study showing that some people have a white blood cell capable of suppressing HIV activity This cell could be producing a protein that counteracts the co-infection effectof Martin's hypothesis.

Other research has pointed to the frequency of AIDS among infected individuals after they were exposed to herpesviruses or hepatitis B. With millions of people infected but not yet showing signs of illness, the problem is more than academic.

But for the moment, what causes infection to develop into AIDS, says Ziegler, 'is a biological black box."

The epidemiologic viewpoint

The first clues about the nature of AIDS came from epidemiology. When epidemiologists chronicled the emergence of the syndrome among male homosexuals, intravenous drug abusers and hemophiliacs, their findings suggested that an infectious agent carried by blood or other body fluids was at work. The biologists eventually found a virus.

Epidemiology has offered no solid leads on whether the virus needs a boost from a cofactor in order to cause disease, nor does it provide clues as to what that cofactor might be. But the suspicion of a cofactor is strong enough to have prompted a search for a common behavioral or lifestyle thread among people with AIDS that is absent in healthy infected individuals.

Studies done to date have not identified any causative cofactors. Epidemiologist Harold Jaffe and his colleagues at the Centers for Disease Control in Atlanta, along with researchers from several San Francisco institutions, are conducting an ongoing study of thousands of homosexual men in San Francisco. Information has been collected about the men's behavior, drug use and health. "So far none of thew has really been predictive of illness," says Jaffe.

Of 104 men in the study who have gone from being antibody-negative, indicating they had not been exposed to the AIDS virus, to being antibody-positive, 15 percent were stricken with AIDS five years after infection. After seven years, 35 percent of the original group had AIDS, but no common behavior or health factor could be identified in the group that became ill "The best predictor of illness seems to be the duration of infection," Jaffe says.

While the study argues against a single obvious cofactor, it doesn't ruled out cofactors entirely- "It might be that there are a variety of things that slightly increase your chances of being sick," says Jaffe. "Instead of one thing increasing your chances by 50 percent, there might be 50 things that increase your chances by I percent, and it's not very likely we'll be able to sort out what those things are.'

Proposed cofactors such as genetic proteins or co-infections identified in laboratory research are not necessarily at work in the real world, says Jaffe. "I have no doubt people have already found and will continue to find factors or agents that will activate the infection in the laboratory," he says. "But you don't know if these things are clinically relevant.

"In a sense I think we want to believe there's something else," says Jaffe, "because if there is something else, it would give us a potential intervention.... We'd like to believe there is such a thing, but so far nobody's found one." -J. Silberner