~MOLECULE OF THE YEAR

NO News Is Good NewsA startlingly simple molecule unites neuroscience, physiology, and immunology and revises

scientists' understanding of how cells communicate and defend themselves

OSt0z A decade ago, nitric oxideE(NO) was just another toxicmolecule, one of a lengthylist of environmental pol-

Molecule lutants found in unsavoryof the Year haunts such as cigarette

smoke and smog. Destroyerof ozone, suspected carcinogen, and precur-sor of acid rain, this gas had a bad reputation.

But over the past 5 years, diverse lines ofevidence have converged to show that thissometime poison is a fundamental player inthe everyday business ofthehuman body.In 1992, NO was ushered into the pan-theon ofmessenger molecules with a fan-fare of hundreds of research papers. Thisyear scientists probed NO's activities inthe brain, arteries, immune system, liver,pancreas, uterus, peripheral nerves, andlungs. They found that the molecule isessential to activities that range from di-gestion and blood pressure regulation toantimicrobial defense.

Nitric oxide also carries importantinformation in the nervous system: Inmales, it is the messenger that translatessexual excitement into an erect penis. In NCthe brain, nitric oxide may be a long- enosought mystery molecule that aids inlearning and remembering.

Yet early wariness concerning the powerof NO was not ill-founded: This year themolecule's derivatives were found to damageDNA in human cells, and it is implicated innerve damage left by strokes.

In 1992, the nitric oxide breeze waftingthrough the scientific world strengthened intogale force winds, stirring up potential drugdevelopment and propeling hundreds ofhere-tofore-unrelated scientists together into a newresearch field.

NO wonder. Not to be confused with itscousin, laughing gas or nitrous oxide (N20),nitric oxide is the smallest, lightest mol-ecule-and the first gas-known to act as abiological messenger in mammals. The mol-ecule has one unpaired electron, making it afree radical that avidly reacts with other mol-ecules. In the presence of oxygen, NO mayvanish a few seconds after it forms, althoughits lifespan in the human body is unknown.No one expected such a bizarre molecule

to be important in physiology, and the earli-est research reports were greeted with disbe-lief. But in the late 1980s, scientists in dis-tinct disciplines-immunology, cardiovascu-

lar physiology, and carcinogenesis-suddenlyrealized they were studying the same mol-ecule. Like a squirt of some powerful per-fume, a puff of nitric oxide spurs differentcells into an array ofdifferent activities, fromcommunication to defense to regulation.

A thousand times NO. In 1992, scientistsprobed the reasons behind these multiple per-sonalities. One significant clue: the biochem-istry of nitric oxide manufacture. Cells relyon various forms of an unusual enzyme calledNO synthase (NOS) to do the job, and a

D trace. Immunohistochemical staining tracks the presIce of NOS and lights up NO-making macrophages.

single cell may have two kinds of enzyme-constitutive and inducible-that produceNOfor different roles. Constitutive enzymes areever-present citizens of the cell, always avail-able to make brief puffs of NO for delicatetasks like neurotransmission. In contrast, in-ducible enzymes are goaded into action moreslowly by other cellular messengers. But overa period of days they can produce at least1000 times more NO for cellular defense. In1992, scientists cloned the genes for a stringofthese enzymatic forms, including constitu-tive NOS from human and bovine endothe-lial cells, and inducible NOS from mousemacrophages and human liver cells.

Understanding this unusual enzyme is cru-cial to designing drugs to turnNO on and off.For example, a handful of molecular cofactorsare tightly bound toNOS and help the enzymeaccomplish its task of stripping five electronsfrom one amino acid (L-arginine) to produceNO and another amino acid. This year bio-chemists discovered that one such molecularhelper is heme, the iron-containing compoundthat carries oxygen and makes blood red. Sinceheme is a known electron acceptor, the newknowledge gives biochemists a start on figur-

ing out how the enzyme works.NO cure for heartache. This year, clini-

cal applications ofNO knowledge bloomedin several directions at once, but much effortfocused on nitric oxide's role as the body'sown blood pressure police. In blood vessels,NO is released by endothelial cells on theinside of the vessel wall, migrates to nearbymuscle cells, and relaxes them. This dilatesthe vessel and lowers blood pressure.

Understanding this process opens the doorto a host of new drugs. Indeed, faults in the

NO system may be the guilty parties insome familiar cardiovascular diseases,

*X possibly even essential hypertension andatherosclerosis. In 1992, scientists racedto develop a new class of chemicals thatcan release NO more controllably andsuccessfully tested them in cell culturesand in mammals.

In a handful ofextreme cases in 1992,physicians have used NO inhibitors tosave lives. Septic shock, a leading causeof death in U.S. intensive-care wards, issomehow related to too much NO: Life-threatening low blood pressure occurswhen the body pours out nitric oxide inresponse to a bacterial infection. Thisyear, nitric oxide inhibitors successfully

brought several patients' blood pressure outof the danger zone in a few minutes. Also thisyear, Phase I clinical trials were begun to testNO inhibitors as an adjunct to interleukin-2(11-2) therapy for stubborn cancers of theskin and kidney. 11-2 rouses the immune sys-tem but also triggers a dangerous flood ofnitric oxide; physician-scientists hope NOinhibitors will keep blood pressure up while11-2 helps kill the cancers.

The power of NO. As a defensive weapon,NO appears to work in at least two ways: byinhibiting key metabolic pathways to blockgrowth, and by killing cells outright. In thefirst mode,NO attacks susceptible iron groupsin certain enzymes, including those that syn-thesize DNA and help cells respire. Whenthose enzymes are crippled, cells cannot growand divide; this may be an important part ofNO's antitumor function.

This year it was shown that NO can alsocombine with oxygen to eventually producepotent cellular assassins such as the hydroxlradical, OH, and nitrogen dioxide. Suchpathways may be behind NO's antibacterialproperties, which are the basis of the centu-ries-old practice of curing meat with nitrite-

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rich salts. In 1992, scientists investigated thepossibility that NO is a primitive defenseagainst a whole range ofmicrobes and workedto understand how host cells defend them-selves against this internal killer.

In the brain, NO's split personality emergesat full strength, as the molecule acts as eithermessenger or killer depending on conditions.For example, evidence gathered this year andlast suggests that neurons con-taining NO synthase may ac-tually kill their neighbors dur-ing a stroke. These NOS neu-rons-themselves eerily resis-tant to stroke damage-appar-ently may become overstimu-lated by the excess neurotrans-mitter released during a strokeand flood nearby cells withtoxic amounts ofNO. In miceand cultured nerve cells, NO NO resistanceinhibitors can reduce damage (gray) live as olsignificantly; scientists are av-idly pursuing the therapeutic implications.NO neurotransmitter. In neuroscience,

startling discoveries ofNO's myriad roles areemerging at a rapid pace. This year nitricoxide has proven itself privy to a host ofdelicate body functions-yet it continues todefy the normal rules of neurotransmission.

This evanescent molecule sometimes doesthe work of a neurotransmitter but doesn'tlook or act like any other such messenger.When a neuron fires, it releases neurotrans-mitter from special storage vesicles into a gapcalled the synapse; the receiving cell picks upthe neurotransmitter and is activated. ButNO has no special storage facilities, and nospecial release mechanisms. Made when andwhere it's needed, it seems simply to diffuseout of the producing cell.

Most neurotransmitters are composed ofamino acids or a string of peptides, whichcouple with precisely configured receptorson the surface of cells. Compared to thismodel,NO is positively promiscuous. It needsno receptor gates, passing through membraneslike a ghost. Its known targets are enzymesdeep within cells, and it carries its message toany and every cell within reach.

NO sex. So what makes this odd moleculeso similar to a neurotransmitter? One of thebest examples comes from sex. This year,scientists proved definitively that in males,NO translates sexual excitement into po-tency by causing erections. Key pelvic nervesget a message from the brain and make nitricoxide in response. NO dilates blood vesselsthroughout the crucial areas of the penis,blood rushes in, and the penis rises to theoccasion. IfNO synthesis is blocked-as wasdone in experiments in live rats this year-those all-important blood vessels never di-late, and the penis stays limp. With 10% ofthe male population suffering from impo-tence, researchers are hotly pursuing this line

t. N)thei

of research in hopes of clinical applications.In addition to being found in the genitalia,

nitric oxide also carries out its duties as neu-rotransmitter throughout the lungs and gut.For example, during digestion, the gut begins aset ofsystematic wavelike contractions-peri-stalsis-that move food through the stomachand intestines;NO triggers the relaxationcom-ponent. This process is blocked in some in-

fants with a potentially lethal_° condition called infantile hy-c pertrophic pyloric stenosis.@ This year, scientists found evi-dence that lack ofNO is theculprit in that disorder.

Learning to say NO. Backin the brain, a series of ex-periments done in late 1991and throughout 1992 suggeststhat nitric oxide may help

JOS neurons cells to store and retrieve in-rs (black) die. formation-the keys to learn-

ing and memory.The cellular basis for learning and memory

is thought to rely on strengthening the con-nection between sending and receiving neu-rons, the presynaptic and postsynaptic cells.One way to do this is a process called long-term potentiation (LTP), in which repeatedfiring causes postsynaptic cells to respondmore strongly the next time they receive asignal. One theory for how it works invokesthe "retrograde messenger," a mysteriousmolecule that would travel backward acrossthe synapse and enhance the release of neu-rotransmitter in the presynaptic cell. Such amessenger would have to pass lightly from cellto cell without benefit of either release ma-chinery or receptors-just like nitric oxide.

So in the past 2 years, neuroscientists haverushed to test nitric oxide. Several labs havenow performed the crucial experiment andgotten positive results: In rat hippocampalneurons, LTP can be prevented by inhibitingNO synthesis. Also this year, scientists in-jected NO inhibitors into the brains of liverats and found that the rats could no longerlearn a water maze.

But not everyone is ready to crown nitricoxide as retrograde messenger. One missingpiece in the puzzle: Thus far, no one has beenable to detectNOS reliably in the hippocam-pal neurons that are supposed to be releasingNO. A few more experimental replicationswill increase everyone's confidence that theeffect is real. Expect intense, focused effort-and a torrent of papers-to resolve this de-bate next year.

NO class? In the brain, nitric oxide sci-ence is in the discovery phase, as neuroscien-tists test NO in a variety of systems. It's fartoo soon to put all the pieces together, butNO is clearly a crucial part of our neuro-chemical system.

Some scientists speculate that nitric ox-ide is merely the first of a soon-to-be-discov-

SCIENCE * VOL. 258 * 18 DECEMBER 1992

ered new class of signaling molecules-gasesthat swiftly pass through a cluster of cells andthen vanish. Preliminary evidence hints thatone candidate for such an airy messengermay be carbon monoxide, another moleculethat today has a shady reputation. Stay tunedfor new developments next year.

Despite this year's burst ofresearch, scien-tists have a long way to go before they under-stand nitric oxide's paradoxical nature. Onpage 1898 of this issue, Stamler et al. arguethat the uncharged, free radical form, NO,may not be responsible for all of the myriadactivities attributed to "nitric oxide." Other,intermediary compounds containing theNOgroup may also play a role. That view is by nomeans a consensus opinion, but the debateitselfbespeaks a new attention to basic chem-istry, especially oxidation and reduction re-actions. As Stamler et al.'s paper illustrates,NO's conceptual contribution has made it-self keenly felt this year. This molecule'sCinderella story-from vaguely noxious gasto powerful queen of communication anddefense-offers a classic example of nature'scontinuing power to surprise.

And the runners-up are...In 1992, science surged forward, often in unex-pected directions. Below are nine advancesthat deserve to share the spotlight-and re-member, they may be composed of billions ofmolecules. Any elegant technique or unusualcorner of the universe may qualify, if it hasearned a place in scientific history this year.

Chromosome countdown. In a landmarkyear for the Human Genome Project, scien-tists in 1992 completed high-resolution physi-cal maps for two of the 24 human chromo-somes, Y and 21. These maps demonstratethat the genome project-which celebratedits second birthday this year-is living up to

New maps. Chromosomes Y (yellow) and 21have been completed.

its promise. The goal of mapping the entirehuman genome by 1995, once consideredoverly ambitious, is now clearly within reach.

Each map is itself a scientific coup. The Ychromosome is the essence of maleness, re-sponsible for dividing our species into two

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HEMaking movies. MRI tthe human visual corte:

manageable chunks,bounded on each side by known sequences.

Also this year, independent groups of sci-entists completed two separate genetic linkagemaps of the whole genome, linking molecularmarkers with inheritance of specific traits.

All this information will speed the workof understanding known genetic diseases, as

well as the genetic components of commonafflictions such as cancer and heart disease.

There's still much to be done, ofcourse-these chromosomes are charted by maps, notby complete sequences. Those going gene

hunting must still do some searching-butnow they know where to look.

Resourceful ribozymes. Ribozymes-RNA molecules that can serve as catalyticenzymes-greatly expanded their known rep-

ertoire of activities this year, showing promiseas therapeutic agents and spurring a flurry ofresearch in both companies and universities.When first discovered, ribozymes' tasks

were restricted to processingRNA molecules.But in 1992, nearly conclusive evidence sug-gests ribozymes can also build bridges be-tween amino acids to form proteins, and can

make and break the bonds that link aminoacids to RNA. This offers a new view of howproteins are made in the cellular structurecalled a ribosome. Biologists once thoughtproteins themselves performed the catalyticlabor, whileRNA merely supplied a support-ive scaffold. This year's results suggest thereverse-the proteins are the scaffolding,and the ribozymes do the work.

Also this year, scientists co-opted thepower of natural selection to coax ribozymesto perform new chemical tricks in the testtube (see article on page 1910). Ribozymes'ability to "evolve" new functions-as well as

their newly expanded catalytic talents-areconsistent with the "RNA world," the theorythat early life relied onRNA to perform manyroles in the biological stage.

The more practical implications of all thisenzymatic activity have not been lost on thebiotechnology industry. Companies old andnew have pounced on the opportunity to

develop specific ribozymes that could slicethe RNA of pathogens such as HIV while

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2 leaving host cells intact.C MRI makes itsF move. Magnetic reso-z

z nance imaging (MRI)' has long offered mul-

tiple benefits to medi-cine and science, be-cause it offers a non-

invasive way to peer in-

*:r1 _ side the body. But in1992, the technologyleaped forward with thedevelopment of func-tional MRI, which al-

,racks changes in lows scientists to trackx as lights flicker. blood flow in the brain

in real time. This year

subjects stepped into a large magnet andstared at lights or squeezed objects, whilescientists made real-time movies of thechanges in their brains. Scientists even toldsubjects to imagine a bright light-andwatched the visual cortex light up.

For years this kind of functional imag-ing-where scientists match thought, deed,or disease to corresponding brain activity-has been the domain of PET(Positron Emission Tom-ography) and SPECT (SinglePhoton Emission ComputerTomography). Now MRI,which does not require injec-tions of tracer elements as

these techniques do, offers a

third alternative.Functional MRI uses mag-

netic fields and radio waves to Seeing parallel

trackchangesinbloodflowand machine modelsblood oxygenation. And it's be-coming quicker and easier all the time. Fansinsist functional imaging doesn't even requirea state-of-the-art MRI machine but could bedone on the 2000 conventional machines inuse in hospitals worldwide. Though Hollywoodneedn't worry about competition just yet, theage of MRI movie-making has begun.

X marks the spot For more than a de-cade, circumstantial evidence has been pil-ing up for an extra-terrestrial villain in one ofgeology's great whodunits: the Cretaceous-Tertiary extinction, when the dinosaurs and

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Heavy hitter. Gravity data outlines theYucatan's 65-million-year-old crater.

many plants and sea-dwelling invertebratesvanished. In 1992, new evidence has broughtone chapter of the story-the search for a

likely impact crater-to a close.Two years ago, geologists honed in on a

giant subterranean crater, 180 km in diam-eter, near the town ofChicxulub in Mexico'sYucatan peninsula. This year, using lasersand high-resolution mass spectrometers to

measure isotopes of argon, two groups of re-

searchers independently dated molten rockfrom the crater at 65 million years ago-rightat the Cretaceous-Tertiary boundary. Samplesfrom Haiti and northeastern Mexico showedthat tektites-beads ofglass forged in the fieryheat of an impact-were exactly the same age

as the crater and similar in chemistry.With data linking impact debris to the

Chicxulub crater, and both to the pivotal$65 million date, many former skeptics nowadmit that something big crashed to Earth at

the end of the Cretaceous era in Mexico.And so the next phase of research has begun:finding out what happened after the crash,and whether more mundane processes suchas climate change served as accomplices in

the death of the dinosaurs.Parallel power. Supercom- w

puter users are accustomed to wOrapidly changing technology.yBut 1992 was something special: Hthe dawn of the era of a new

style ofsupercomputer, the mas-sivelyparallel machine. This year Onearly a dozen supercomputer mcompanies introduced or an-

A parallel nounced plans for massively par-water. allel computers. Scientists used

these machines to tackle an ar-

ray of complex problems, from imaging thesurface of a silicon chip to deciphering thestructure of an ion channel protein.

Most of today's supercomputers use more

than one processor-the Cray Y-MP haseight, for example-but massively parallelmachines represent a new type of computerarchitecture, in which a small army of pro-cessors can dissect and work huge problemsat awesome speeds. Devotees foretell a thou-sand-fold increase in peak computer speed inthe next few years.

What kinds of science will these new ma-

chines solve? Any problem rich in data andcomplexity-the "grand challenges" of sci-ence, including the mysteries of molecularinteractions, protein folding and structure,properties of materials, climate change, andthe evolution of galaxies.

Bench scientists can take heart in anotherlandmark this year: jumps in the speed ofcomputer communications. Today scientistscan communicate on Internet, with eachother and with supercomputers, at speeds upto 45 million bits per second, a goal achieved1 year ahead of schedule. Next year, expectspeeds of 155 million bits per second.

SCIENCE * VOL. 258 * 18 DECEMBER 1992

mSuC

sexes. Chromosome 21has been linked to ge-netic disorders such asDown's syndrome andfamilial Alzheimer's.

To map the chromo-somes, scientists clonedoverlapping pieces ofDNA, each tagged withone or more known se-quences, and then putthe tags in order. Thusthe vast stretches of un-known DNA that makeup the chromosomeshave been divided into

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Nitrogen news. One of the triumphsof the Industrial Revolution was the fHaber process, which allows chemiststo create ammonia, NH3, from nitro-gen gas, N2, by using strong-arm tacticsofhigh temperature and pressure. But sci-entists have long known that nature does itbetter. Certain bacteria in the roots of plants

Z perform the same reaction at atmospheric pres-sure and temperature, in the process makingnitrogen available to other life forms.

How do these bacteria do it? Nineteen-ninety-two saw the start of the era in whichchemists and biologists can begin to answerthat question. This year, scientists revealedthe structure of nitrogenase, the enzyme re-sponsible for the enviable abilities of nitro-gen-fixing bacteria.

During the past de-cade, two independentgroups ofscientists havecarried out the painstak-ing work needed to de-duce the enzyme's com-plex structure. Their re-sults show that nitroge-nase is essentially twocomplicated proteins,an iron protein and aniron-molybdenum pro-tein, which work to-gether to catalyze the re-action. One surprise wasthe molybdenum: chem-ists had long thoughtthis transition metal Nitrogenase unveilcrucial to the catalytic bottom, and Mo-Fe,process. But the molyb-denum atoms turn out to be so thoroughlytied up with other bonds that they seem un-likely candidates for the actual site of nitro-gen reduction.

Finding the structure set offa flurry oftheo-rizing-and experimentation-by chemistswho think they can already see how electronsmight be persuaded to take the crucial steps:from the iron protein, to the molybdenum-iron protein, and finally to N2. However theelectrons make their journey, the structure ofnitrogenase has begun a new age of explora-tion for a new generation of chemists.

Space tracks. Cosmology, a field rich intheory but poor on experiment, this year re-ceived a rich inoculation ofdata from a satel-lite seeking the seeds of galactic structure.Called the Cosmic Background Explorer(COBE), the National Aeronautics andSpace Administration's probe was sent outto take the temperature of the early universeby measuring the faint afterglow left by theBig Bang.

The occurrence of that primordial explo-sion is backed up by pervasive microwaveradiation, which bathes the universe at ex-actly the temperature predicted by Big Bangtheorists, 3 degrees above absolute zero.

Cosmic kernels. COBE found traces of thestructure of the early universe.

led.top

But Big Bang theory also suggests that 15billion years ago the newborn universe was afeatureless structure, stretching out evenly inall directions. Today matter is clumped tightlyinto galaxies like our own Milky Way, withvast reaches ofempty space in between. How

did galaxies and star clus-W ters emerge? Cosmolo-; gists had predicted slight

E) bumps in the texture ofthe early universe, whichover the eons werestrengthened by theforce of gravity into ma-jor cosmic structures.Traces of these early ir-regularities should bepreserved today as slightvariations in the back-ground radiation.

In 1992, after manymonths of orbit, COBEgot- the goods: It mea-

Two proteins (Fe, sured slightly cooler and) work together. warmer spots, differing

by only 30 millionths ofa degree, in the cosmic microwave radiation.Thus cosmologists' notions of the early uni-verse have been vindicated. More good newsarrived this month, when data from a balloon-borne experiment, which scanned a smallerarea of sky at a different wavelength, partiallyconfirmed COBE's results.For cosmological explorers,looking up has paid off.Common antisense.

Scientists have long recog-nized the potential powerof knocking out specificgenes with antisense mol-ecules. This year, thisbenchside technique blos-somed into drug develop-ment, and antisense com- The youngest ppounds were used in hu- medicine forgesmans for the first time.

Antisense molecules prevent cells fromtranslating genetic information into proteins.Nucleic acids have two complementarystrands, only one of which-the "sense"strand-codes for protein. The other strandis "antisense," which can bind to sense mol-ecules and prevent them from being ex-pressed.

pati,ah

Antisense weapons have been craftedto fight leukemia, AIDS, hepatitis, andother diseases. This year scientists used avirus to slip antisense RNA moleculesinto cells already infected with HIV. The

results: HIV infectivity decreased by morethan 99% in vitro.Another antisense technique relies on

relatively short synthetic DNA molecules(called oligonucleotides) that are apparentlyspontaneously taken up by cells, and so needno special means of delivery. This year, thefirst human trials of these compounds began,in a patient with leukemia and in patientswith human papilloma virus, which causesgenital warts. And in September, the Na-tional Institutes of Health approved the firstantisense gene therapy trials, scheduled tobegin next year in patients with lung cancer.

Embryonic efforts. In 1992, medicinemarched backward to the earliest stages oflife, breaking new ground in fetal diagnosisand treatment and gaining greater under-standing of the biology of life in utero.

This year, the healing power offetal tissuewas used to treat Parkinson's disease in ahandful of studies. The results were encour-aging-several patients regained their abil-ity to walk and talk. But transplanting fetaltissue to adults is only the beginning. Be-cause the fetal immune response is not yetfully developed, undeveloped fetuses them-selves may be ideal recipients for transplants.In 1992, scientists continued to explore thepotential for fetal medicine in animal mod-els, and in some cases performed surgery tocorrect anatomical defects in utero.

In fetal diagnosis, testing was pushed backto the first few days of life in a procedureappropriately nicknamed BABI (BlastomereAnalysis Before Implantation). Coupled within vitro fertilization and performed at theeight-cell stage, the technique allows physi-cians to screen embryos for specific genetic

diseases before implanting< them into the mother's

uterus. In 1992, BABI pro-duced a healthy baby girlwho does not carry the genefor cystic fibrosis-eventhough both her parents do.

Scientists also opened anew diagnostic frontier thatmay eventually allow fetaltesting simply by screening

ients. Fetal the mother's blood. Newead. techniques can isolate fetal

cells from maternal cells inthe bloodstream, and in two cases have iden-tified fetuses with major genetic defects.

Taken together, these developments pavethe way for a new kind of medicine, in whichpatients are diagnosed-and treated-months before they are born.

-Elizabeth Culotta andDaniel E. Koshland Jr.

SCIENCE * VOL. 258 * 18 DECEMBER 1992 1 865

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