Gene Expression in the Aging Brain

1512 normal enzyme. Then, they replaced the oxygen in question with a sulfur atom. The reaction didn’t work as well because, by the rules of chemistry, sulfur doesn’t like to bind to magnesium. But sulfur does like manganese and cadmium ions. So they replaced the magnesium with one of these other metal ions and measured the reaction. The researchers saw that these other metal ions restored (or “rescued”) enzymatic activity. In short, the enzyme needs a bond where the oxygen and the magnesium are, but the bond doesn’t have to be between oxygen and magnesium. As complicated as that is, plucking out one atom and trading it for another is itself a tricky business. Because most enzymes are made of stubborn amino acids and not nucleotides, atomic mutagenesis can be diffi cult. And usually when researchers have tried atomic mutagenesis, they’ve mutated the substrate (the molecule that the reaction acts upon) instead of the enzyme (the molecule that acts). Here, Piccirilli, Herschlag, and colleagues directed the applications of atomic mutagenesis to the molecule that does the work. To test that a specifi c oxygen in the intron binds to the magnesium ion, the researchers fi rst had to compile a short list of potential atoms to which the magnesium might bind. By combining literature data from structural models and functional studies with a random sprinkling of sulfur atoms in the intron to fi nd critical oxygen contacts, Piccirilli, Herschlag, and colleagues established a group of specifi c oxygen atoms to watch. They tried the metal rescue experiment with each of these oxygens, and the only enzyme rescued by the metal switch was the one in which they changed the C262 oxygen to a sulfur. Therefore, they concluded that this specifi c oxygen atom makes a critical contact with the magnesium ion. The strategy of atomic mutagenesis combined with metal ion rescue can be used to help understand the mechanism of other RNA and protein metalloenzymes.

normal enzyme. Then, they replaced the oxygen in question with a sulfur atom. The reaction didn't work as well because, by the rules of chemistry, sulfur doesn't like to bind to magnesium. But sulfur does like manganese and cadmium ions. So they replaced the magnesium with one of these other metal ions and measured the reaction. The researchers saw that these other metal ions restored (or "rescued") enzymatic activity. In short, the enzyme needs a bond where the oxygen and the magnesium are, but the bond doesn't have to be between oxygen and magnesium.
As complicated as that is, plucking out one atom and trading it for another is itself a tricky business. Because most enzymes are made of stubborn amino acids and not nucleotides, atomic mutagenesis can be diffi cult. And usually when researchers have tried atomic mutagenesis, they've mutated the substrate (the molecule that the reaction acts upon) instead of the enzyme (the molecule that acts). Here, Piccirilli, Herschlag, and colleagues directed the applications of atomic mutagenesis to the molecule that does the work.
To test that a specifi c oxygen in the intron binds to the magnesium ion, the researchers fi rst had to compile a short list of potential atoms to which the magnesium might bind. By combining literature data from structural models and functional studies with a random sprinkling of sulfur atoms in the intron to fi nd critical oxygen contacts, Piccirilli, Herschlag, and colleagues established a group of specifi c oxygen atoms to watch.
They tried the metal rescue experiment with each of these oxygens, and the only enzyme rescued by the metal switch was the one in which they changed the C262 oxygen to a sulfur. Therefore, they concluded that this specifi c oxygen atom makes a critical contact with the magnesium ion. The strategy of atomic mutagenesis combined with metal ion rescue can be used to help understand the mechanism of other RNA and protein metalloenzymes.

Synopses of Research Articles
Open access, freely available online September 2005 | Volume 3 | Issue 9 | e310 | e317 Ribonucleic acid (RNA) is a dogma breaker. The "central dogma" of cellular biochemistry mandates that deoxyribonucleic acid (DNA) stores information, and RNA copies this information and uses it to direct the assembly of amino acid building blocks into proteins, such as enzymes. Enzymes catalyze important chemical reactions in the cell, such as the breakdown of glucose or the synthesis of urea.
When biochemists discovered catalytic RNA, they had to ditch the dogma. Because of its structure, it turns out, RNA can act as an enzyme and catalyze reactions. While two strands of DNA tend to zip up into the famous double helix, RNA usually goes solo. The single RNA strand folds back on itself to create myriad tangled arrangements. Some of these arrangements create an active center, the place on the RNA where the enzymatic magic happens. The many RNA enzymes and protein enzymes that use metal atoms to do their job are called metalloenzymes. One example of an important structural motif in RNA metalloenzymes is the group I intron, which can snip itself out of an RNA segment. Understanding exactly how the RNA and the metals interact will help to provide precise answers about how the enzyme really works.
Through X-ray crystallography, researchers have revealed many structural features of group I introns. But X-ray crystallography creates images of the enzyme frozen in time; it does not catch an enzyme in action. In a new study, Joseph Piccirilli, Daniel Herschlag, and colleagues discovered that a particular oxygen atom on a particular nucleotide in a group I RNA must bind to a particular magnesium ion in order for the reaction under study to proceed normally. The oxygen atom is known as the pro-S P phosphoryl oxygen at nucleotide C262 in the intron from the unicellular Tetrahymena thermophila protozoan.
Since there's no way to watch the oxygen and metal hook up during the reaction, how do the researchers know they do? The researchers used the powerful techniques of metal ion rescue and atomic mutagenesis. Here's how it worked. They fi gured out how well the group I intron reaction works with a The Ol' Switcheroo Shows How an RNA Enzyme Splices Itself DOI: 10.1371/journal.pbio.0030310 DOI: 10.1371 RNA enzymes called ribozymes require metal atoms to function. The site of metalribozyme interaction was studied by changing components of both the catalytic center of the ribozyme (the backbone) and its target substrate.
As warm-blooded creatures, humans and other mammals maintain a relatively stable body temperature that buckles under the stress of intense heat (or cold). When the heat gets too high, we develop fevers and weaken, and our proteins destabilize and degrade-in some cases, such reactions can prove fatal. But some organisms appear to defy nature (as we think of it) by fl ourishing in extremely high temperatures. The archaeal microbe Pyrobaculum aerophilum, for exampleoriginally found in a boiling marine water hole in Italy-thrives at ~100 °C (212 °F). Similarly, the bacterium Thermus thermophilus grows at temperatures between 48 °C and 85 °C (118-185 °F).
Such organisms are of interest for many reasons-not least of which is to understand the mechanisms that engineer their heat resistance, or thermostability. How do these thermophilic bacteria and archaea manage to maintain active, stable proteins at such high temperatures? In an elegant demonstration of how the Genomics Helps Explain Why Some Like It Hot DOI: 10.1371/journal.pbio.0030317 ever-growing publicly available genome sequence and protein structure data can be analyzed, Todd Yeates and colleagues identify one answer to this question.
The authors found that proteins from P. aerophilum and T. thermophilus, along with some other thermophiles, have many disulfi des, which are known to improve stability. Disulfi des are covalent bonds that form when the sulfhydryl groups (a sulfur and a hydrogen atom) of two spatially proximate cysteines (one of the 20 amino acid building blocks of proteins) are oxidized. When conditions are right, the two hydrogen atoms are removed by other molecules in the cell dedicated to that purpose, and the remaining sulfur atoms form a bond.
The authors mapped sequences of intracellular genes from 199 prokaryote genomes onto sequence-related proteins with known three-dimensional structures. The resulting structural models reveal when disulfi de bonds are likely to form. A pronounced bias was found for disulfi des in a set of thermophilic genomes. To prove that these predictions really do form disulfi de bonds, the authors solved the structure of one protein from P. aerophilum-which was indeed stabilized by three disulfi de bonds.
Disulfi de bonds form more commonly outside or between cells in multicellular organisms, where the environment is ideal for two cysteines to cozy up and bond in an oxidative extracellular location. The high numbers of bonds observed in these single-cell prokaryotes not only help explain thermostability but also challenge our ideas of how disulfi de bonds form. Given the presumed diffi culty for disulfi des to form in such organisms, the authors set out to look for any proteins that might help explain the mystery. They investigated which proteins are present in the disulfi de-rich organisms as compared with the proteins in other organisms (also known as phylogenetic profi ling). The authors discovered that all of the disulfi de-rich thermophiles had something else in common: they all encode a protein not seen in other organisms, called protein disulfi de oxidoreductase (PDO). As its name suggests, this protein likely plays a key role in the formation of disulfi des in these heat-tolerant bugs.
Yeates and colleagues have considerably advanced our understanding of how proteins withstand and remain functional at high temperatures in these thermophilic organisms (via additional stabilizing disulfi de bonds). Yet, since this correlation of extra disulfi des and the PDO is not common to all thermophiles, it seems likely that this is not the only method employed in heat resistance. Probably a fi nely tuned concert of different mechanisms works in synchrony to enable thermophiles to fl ourish in extreme conditions. As the authors show here, it's likely that genome sequence and structure data can help us to uncover these mechanisms. Have you ever wondered how the myriad insect forms-beetles, fl ies, dragonfl ies, mosquitoes, grasshoppers, ants, wasps, bees, and countless others-evolved? Insects make up 75% of all species known. The large number of insect species is probably a result of a combination of one or more factors: a high rate of formation of new species, or speciation, an ability to adapt to new environments and exploit new ecological niches, and a lower rate of extinction. Speciation, adaptation, and extinction are all controlled by the interplay between genetic and environmental factors. Understanding the genetic changes that lead to the formation of new species is an important area of research in evolutionary biology.
In a new study, Thomas Turner, Matthew Hahn, and Sergey Nuzhdin worked with the malaria mosquito Anopheles gambiae to uncover genes that may be driving speciation. A. gambiae exists in multiple forms that may be in the early stages of differentiating into separate species; on the other hand, they may be partially differentiated, co-existing races that could give us valuable information on genes responsible for racial differences in mosquitoes. Turner and colleagues focused on two forms, A. gambiae M and A. gambiae S, that sometimes mate and create hybrid forms in nature. While it's unclear whether the forms can produce fertile hybrid offspring in the wild, the progeny of lab matings appear to have no problems with fertility. This suggests that individuals either naturally prefer to mate with others of their own form, or that there must be environmental and/or genetic conditions that are not favorable for the survival of hybrid progeny in nature.
To study the genetic underpinnings of speciation, the researchers used DNA microarrays to identify global differences Islands in the Genome Promote Speciation in the mosquito genomes. Using a combination of gene chips, statistics, and computational biology, Turner and colleagues found that the M and S genomes differ at just three regions. The researchers suggested that genes present here may be responsible for early speciation. These three "speciation islands" in the genome contain 67 predicted genes. In a preliminary analysis of seven of these genes, Turner and colleagues identifi ed fi ve that are different between the two Anopheles forms; these include genes that play a role in a range of cellular processes, including energy metabolism, response to sudden increases in temperature (heat shock), and ion transport across cell membranes. Future work focusing on the 67 genes hypothesized to reside in the divergent regions should yield interesting clues to the identity of genes that drive speciation, and the mechanism by which they do so. This is a signifi cant fi nding in the fi eld of speciation research: in terms of methodology, this study shows that DNA microarrays can be used to identify regions of the genome that are different between two diverging species, allowing researchers to home in on potentially interesting genes. This study also shows that in spite of possible cross-fl ow of genetic material (natural hybrids between the two forms are found at a low frequency) between two populations, the populations can still be accumulating differences in their genomesdifferences that could eventually lead to the formation of new species. Comparing results in Anopheles and the well-studied insect model Drosophila, in which scientists have also started identifying "speciation genes," should tell us if similar genes are employed repeatedly in different genera during the formation of new species. The coil of a snail shell can be either right-handed (dextral) or left-handed (sinistral), based on whether the shell spirals out clockwise or counterclockwise when viewed from above. Most species are composed entirely of individuals that are one or the other type; in exceptional cases, populations may differ in their handedness, or chirality, but within a single population, all individuals tend to be alike. This makes sense, since the mechanics of reproduction are harder between two individuals of opposite chirality (their genitalia are also reversed), reducing the likelihood that they will successfully mate and produce offspring. Over time, therefore, the rarer type will become rarer and rarer until it goes extinct.
This poses the interesting evolutionary question of how a species of one chirality can give rise to another of opposite chirality. If the rarer types are less likely to reproduce, then how do they ever establish themselves beyond a threshold frequency? If they are able to establish themselves, then is a change in chiralitywhich is caused by a single geneenough to isolate them so that they are a new species? A study in this issue by Angus Davison et al. sheds light on the complex interplay of factors that infl uence evolution in the snail Euhadra. Although a single gene does cause a change in chirality, and snails with different chirality are able to mate only with great diffi culty, there is nevertheless almost free gene fl ow between them. Other factors must ultimately become involved to cause speciation.
The 22 species of Euhadra are landdwelling natives of Japan, and include fi ve sinistral and 17 dextral species. Using mitochondrial DNA analysis to construct a family tree, the authors showed that the sinistral species compose a distinct branch, indicating that this feature arose only once in the history of the genus. How did the fi rst sinistral shell types arise, and why didn't they gradually evaporate from the gene pool? One possibility is "reproductive character displacement," in which a new feature that directly affects mating, such as sinistral shell chirality, decreases the likelihood that its owner will mate with snails of other, closely related, species that live nearby. While their dextral brothers or sisters waste valuable resources in such unsuccessful interspecifi c pairings, the few sinistral individuals engage in fewer, but more productive, matings exclusively with their own kind, thus increasing their numbers despite the odds stacked against them.
To test this hypothesis, Davison et al. constructed a model that took into account a variety of factors, including population density, the proximity of other species, and the maternal inheritance pattern of shell chirality (the direction of a snail's shell is determined so early in development that it is governed not by its own genes, but by its mother's). The surprising conclusion is that the last factor, the unusual mode of inheritance, allows for near free gene fl ow between the two forms within a population, even if the two forms are themselves almost unable to mate. The reason is that the offspring of a sinistral mother could itself be sinistral, even if it contains entirely dextral genes. Its offspring, though, might include dextral snails, because its own dextral genes determined their shell chirality.
Their model indicated that new chiral types are able to arise, in spite of there being fewer suitable mates, if there is reproductive character displacement. They cannot be considered new species, however, because of the gene fl ow between them. Reproductive character displacement can account for the speciation of sinistral Euhadra only under a complex set of conditions. Interspecifi c mating would need to be common among the dextral snails. High population density helps, since it allows those with the rare new form to fi nd each other more easily. But gene fl ow between left and right forms would preserve the population as a single species, unless other factors, such as difference in habitat use or geographic separation, increased the isolation of the two forms. This argues against so-called "single-gene speciation," and shows that the creation of a new species requires more than a simple twist of fate. Last October, as Americans started lining up for fl u shots, news broke that 48 million vaccine doses had been contaminated. With 100 million people considered at high risk and fears of a potentially deadly avian fl u epidemic on the horizon, the shortage caused long lines, allegations of price gouging, and a new bill to bolster the nation's anemic vaccine manufacturing base.
Infl uenza A viruses are RNA viruses that infect humans, pigs, horses, and birds, both wild and domestic. Flu infection relies on a viral glycoprotein, hemagglutinin (HA), that binds to receptors on a host cell and allows the virus to be internalized. If antibodies produced by host immunity recognize viral antigens (on the surface of the HA protein), HA binding is inhibited and infection prevented. A virus's best chance of gaining the upper hand in this evolutionary game of cat and mouse is to change its HA in a way that eludes antibody recognition. Typically the mutations are minor and the virus's antigens conserved enough for the host body's immune system to recognize them. On occasion, infl uenza can acquire an antigenically novel HA subtype, becoming a virulent pandemic strain that completely escapes immune surveillance and kills millions. Minimizing the effect of yearly infl uenza outbreaks-by developing effective matched vaccines-depends on predicting which fl u strains are likely to evolve.
Toward this end, Eddie Holmes and colleagues took the global approach afforded by genomics to explore the forces underlying viral adaptations. In all the hullabaloo about stem cells, nobody has noted their uncanny similarity to pizza dough. You can divide either into two or four or eight identical pieces, but that doesn't determine what kind of cell or pizza you're going to make. But once you let a cell grow hundreds of nuclei, or you pile on the pepperoni, you're on your way to making a skeletal muscle fi ber or a pepperoni pizza. If you want a white blood cell or an all-veggie pie, you're out of luck. The commitment to becoming a certain cell type is called differentiation.
Stem cells in living organisms can multiply without differentiating, preserved by molecular signals in special niche environments; without these signals in the petri dish, they differentiate. Pluripotent mouse embryonic stem (ES) cells, a special type of stem cell with the potential to develop into many different cell types, are an exception. Because they divide symmetrically, the scads of artifi cially grown ES cells are all the same. This leads researchers to wonder: what conditions in the body keep stem cells from differentiating, why are ES cells the only kinds that don't differentiate in the petri dish, and how can scientists create undifferentiated tissue-specifi c stem cells in the lab?
In a new paper, Austin Smith and colleagues developed a method to produce symmetrical divisions of mouse brain stem cells derived from ES cells. Their novel method creates an on/off switch for differentiation of tissue-specifi c stem cells: they can multiply without differentiation, and they can also become normal brain cells. The authors also managed to cultivate the brain stem cells without re-creating the rarefi ed neurosphere, the highly specialized environment or microenvironment in which the body grows its own brain stem cells.
Many scientists believe that in the body, these microenvironments prevent stem cells from differentiating. Neurospheres, for example, contain some undifferentiated brain stem cells fl oating in a broth of differentiating cells. One feature of the neurosphere is that a very low percent of cells are brain stem cells. In fact, neurospheres have so few of these cells that scientists have a hard time even observing them. But by cultivating brain stem cells outside the neurosphere, the scientists showed that a complex microenvironment may not be necessary. To grow their stem cells, Smith et al. combined epidermal growth factor (EGF) and fi broblast growth factor (FGF), two small proteins that bind to stem cells and promote growth.
Previously, scientists had grown brain stem cells with FGF. Upon removing FGF, the cells failed to differentiate and become mature. The cells that Smith et al. grew, in contrast, became mature cells upon removal of the growth factor cocktail. They observed both neurons and astrocytes, the two types of cells into which the brain stem cells mature.
In the future, scientists may use this new technique to produce large quantities of the cells to study their basic properties and also to explore their value for modeling neurodegenerative affl ictions, including Huntington disease, Parkinson disease, and Alzheimer disease. Additionally, these cells may clinch the debate of whether doctors will be able to use stem cells directly to repair brain damage. Insulin or insulin-like proteins signal developing animals to grow. After a meal, the body creates insulin, allowing an organism to grow and compete with other organisms for available food. When food is scarce, insulin levels remain low. Only small organisms with low metabolic needs will survive the potential famine. Scientists can study how genes involved in insulin signaling affect development by mutating a gene and seeing what happens to the adult. This useful method, called gene knockout, provides insight into the specifi c relationship between a gene and its physical manifestation, or phenotype. By using the knockout method, scientists can observe how the growth of an organism responds to fl uctuations in insulin signaling levels.
In a new study, Alexander Shingleton and colleagues used a temperaturesensitive mutation in an insulin-receptor gene to discover how alterations of insulin signaling in the fruitfl y Drosophila affect different stages of fl y development. At one stage, the researchers discovered, insulin signaling infl uences total development time, at another it infl uences body size, and at a third stage, it infl uences only organ size.
So when do developing fl ies need insulin? The researchers found that low insulin signaling during very early development extends total development time. Then the larvae reach their critical size, the watershed moment in insect development when larvae commit to becoming pupae. After critical size, reduced insulin signaling no longer delays development but instead leads to petite fl ies with petite organs. When the larvae become pupae, however, reduced insulin signaling simply creates smaller organs. Because developmental time, body size, and organ size each display different responses to reduced insulin signaling activity, these features may evolve independently, the authors reasoned.
Shingleton and colleagues used fl ies with mutant insulin-receptor genes whose protein products are partially inactivated at high temperatures. circulating in the population at the same time, and a more complex evolutionary pattern than previously thought. They also showed that co-circulating viruses can exchange genes in a way that creates antigenically novel, epidemiologically signifi cant strains-a process that humans may facilitate by simultaneously hosting more than one strain.
They analyzed the genomes of 156 infl uenza A viruses (serotype H3N2) collected by New York State public health offi cials between 1999 and 2004 in search of global patterns of viral evolution. Using the fl u virus genome sequences produced at the Institute for Genomic Research (TIGR), funded by a National Institute for Allergy and Infectious Diseases (NIAID) initiative, the authors grouped the viral sequences according to sequence similarity. They also included partial fl u sequences obtained from other studies in their analysis. These data are the initial output of the fi rst large-scale effort to completely sequence infl uenza genomes. While most of the virus genomes sampled after 2002 fell into one group-which the authors called clade A-there were also other clades circulating at different times (called clades B and C).
Gene trees, or phylogenies, constructed for each of the virus's eight genes all diverged according to their respective clades, except one-the HA gene. The HA gene cluster grouped all the clade A viruses that emerged after 2002 as well as both the clade B and C viruses from the same time period and viruses from multiple locations (in Asia, Australia, Europe, and North America).
Altogether, these results indicate that different viral strains had circulated in the same populations until 2002 and then the clade A and C viruses acquired a common HA gene from clade B through reassortment. While reassortment between co-circulating human infl uenza strains has been previously described, this study is the fi rst to examine in detail a reassortment event leading to an epidemiologically signifi cant outcome, the emergence of the "Fujian" strain in the 2003-2004 season. Though it's not yet clear how variant clades manage to persist alongside dominant strains, the fact that they do suggests the infl uenza virus has multiple adaptive tools at its disposal. Luckily, the tools of genomics should help predict what evolutionary paths the virus might take and help in the process of selecting the most promising vaccines to contain it.

A transmission electron micrograph of the infl uenza A virus. New evidence suggests that fl u viruses can rapidly reshuffl e genetic material and mutate into new strains capable of widespread infection. (CDC/Dr. Erskine Palmer)
Malaria is a misnomer. People used to believe that poisoned or "bad air," the translation of the Italian phrase "mal aria," caused disease. In the 19th century, when parasitologists fi gured out that single-celled parasites cause malaria, they didn't bother to change the disease's name. Experimenters proved that these parasites need a host organism to survive-so they can't be transmitted through air-and that the hosts, mosquitoes, carry the parasite to humans. Researchers were optimistic that if they could fi nd a disease's cause, they could also fi nd the cure. Kill the mosquitoes and eradicate malaria. And with the advent of DDT and less environmentally harmful insecticides, potent anti-malarial drugs, and international funding in the late 20th century, eradication of malaria seemed imminent.
But that expectation underestimated the fl exibility of living creatures. Mosquitoes acquired resistance to insecticides while the parasites acquired resistance to anti-malarial drugs. Worse, the aggressive eradication campaign skipped over vast regions of the globe, especially sub-Saharan Africa.
Malaria remains a devastating problem in Africa for several reasons. Environmental conditions provide an amenable atmosphere for both Plasmodium falciparum, the most dangerous form of the parasite, and the Anopheles gambiae mosquito, the most effective vector. Also, many countries in sub-Saharan Africa lack the infrastructure to protect their citizens from malaria. Given the overwhelming scope of malarial infection in Africa, new understanding of the disease will help epidemiologists devise targeted antimalarial strategies.
A new study conducted in Western Kenya by Jacob Koella and colleagues analyzed mosquito behavior to discover how it facilitates the transmission of malaria. The research determined that mosquitoes are more attracted to people infected with transmittable malaria than to either people infected with non-transmittable forms of the disease or uninfected people. To measure the attraction of the mosquitoes, the researchers set up a chamber of infected mosquitoes surrounded by tents containing the study participants. A device called an olfactometer wafted the odors of each participant toward the mosquitoes. Researchers measured which smell most attracted the hungry bugs.
This question had long stalled scientists because of contradictory and indirect evidence. Sweat, breath odor, and high body temperature all increase mosquitoes' blood lust, and no previous study had isolated the variable of malarial infection.
To control for the natural variation in how attractive mosquitoes found each participant, Koella et al. compared the number of mosquitoes that were attracted to infected people to the number of mosquitoes that were attracted to those same people after they were no longer infected. The researchers found that in general, an individual attracted more mosquitoes when infected with transmittable malaria. This demonstrates that malaria, in addition to causing fever, vomiting, headache, and sometimes death, causes more mosquito bites. The biting mosquitoes will then pick up the parasite and spread it to other people.
As another control, the researchers compared infection with a nontransmittable form of the parasite to infection with the transmittable form and to no infection. A mosquito can pick up the malaria parasite only when in its sexually reproductive stage. The transmittable parasite, known as a gametocyte, multiplies in the mosquito's belly before traveling to the mosquito's salivary glands and, eventually, to the blood of the next human victim. But the malaria parasite has a complicated life cycle that also includes non-transmittable asexual stages. Koella and colleagues found that these parasitic forms, unlike the sexually reproductive form, did not make humans more attractive to mosquitoes.
Previous to the recent study, malaria researchers had proved that mosquito biting rates greatly infl uence the spread of malaria. Koella and colleagues showed that the parasite itself increases these biting rates when it is ready for a new host. watched what happened to their bugs. Using this temperature-sensitive insulinreceptor gene, the researchers found that, besides affecting development time, insulin signaling also plays a role in the differential growth rates of different organs. By tracking three organs on male fl ies, Shingleton and colleagues discovered that the genitals are less sensitive to reduced insulin signaling than either the wings or the maxillary palps, olfactory components of the mouth. The authors also found that insulin signaling affects cell size and cell number differently. While slightly reduced insulin signaling shrinks cell size, highly reduced insulin signaling lowers cell number without affecting cell size. By incorporating the effects of reduced insulin signaling into the Drosophila development process, the authors constructed a model of Drosophila development that explains the various roles played by the insulin-signaling pathway during development.
Because the new study alters genes during development, it provides the details of when and how a developing animal requires insulin. Future fl y studies may reveal why organs have individual responses to insulin signals, what other signaling pathways play a role in development, and how insulin came to infl uence so many different features of the developing fl y at different times. To control what you eat and when, your nervous system must coordinate a laundry list of signals: internal signals contain information about energy level, food preferences, and metabolic need, while external signals relay information about the quality of available food, determined by its smell and taste. Scientists studying the fruitfl y Drosophila have traced the path of olfactory signals beginning with chemical receptors in the mouth, which set off neurons that signal the antennal lobe of the central nervous system. From here, the electrical stimulation zooms toward the so-called mushroom body, a mushroom-shaped cluster of neurons involved in olfactory processing. Less is known about the gustatory signals, which begin both in the mouth and in the pharynx and aim toward the subesophageal ganglion region of the fl y's brain. How olfactory and gustatory signals infl uence feeding patterns remains murky.
In a new study, Michael Pankratz and Christoph Melcher used genetic analysis to gain insight into the adult and larval neural networks that use taste information to regulate eating. Specifi cally, they found that several types of neurons responsible for coordinating taste signals express the gene hugin (hug), a gene linked to abnormal eating activity and expressed in only the subesophageal ganglion. By altering hug expression, the researchers uncovered the gene's behavioral infl uence: hug-expressing neurons infl uence a fl y's decision to sample new food sources. The researchers also proposed that hug proteins play a role in hormone-triggered growth, an important consequence of adequate feeding.
To begin their investigation, Melcher and Pankratz analyzed the DNA from fl ies with abnormal eating behavior. One group of these fl ies shared a mutant klumpfuss (klu) gene, normally responsible for encoding a protein transcription factor. Because neural transcription factors control production levels of other neural proteins, the researchers used DNA microarrays to compare gene expression in normal fl ies to that in klu mutants. Any klu-controlled genes expressed at different levels in klu mutants might contain clues about the neural circuitry modulating feeding behavior.
Using microarrays, Melcher and Pankratz discovered that mutant fl y larvae overexpress the hug gene, which is known to encode at least two neural proteins related to growth signaling. The researchers then investigated which signals infl uence hug expression by exposing larvae to either high or low food levels. Because both starved and sugar-fed fl ies express little hug, the researchers inferred that hug levels do not solely signal internal energy requirements but respond to internal and external signals carrying information about the quality of food. The researchers also noted that the fi nicky pumpless (ppl) mutants, which have a feeding defect similar to klu, overexpress hug.
Behavioral studies confi rmed that too much hug reduces food intake and leads to stunted growth, while too little stimulates eating. Melcher and Pankratz selected a group of fl ies and blocked the synapses of their hug neurons to inhibit the neurons' activity. In contrast to control fl ies, which start feeding on a novel food source only after an evaluation phase (they wait a while before initiating feeding), the experimental fl ies started eating new food right away. These hug neurons may help fl ies decide whether or not to eat a new food source.
Larvae express hug in only about 20 neurons, all located in the subesophageal ganglion. The axons of some of these hug neurons extend into the ring gland, a crucial metabolism and growth organ in fl ies. Other axons contact the protocerebrum, a structure close to brain centers for learning and remembering odors. A third set of these axons extend to throat muscleswhich is surprising because most subesophageal ganglion neurons have no connection to motor function. All together, these few hug neurons can signal structures controlling growth, feeding, and learning and memory.
Besides linking hug neurons to brain centers that regulate taste-related feeding behavior, the study also raises questions about how the nervous system prioritizes internal and external signals. How hungry must fl ies be to overcome taste aversion? How do the competing neural networks of taste and hunger signals decide whether the fl y will eat? Future studies pairing behavioral and genetic analysis may begin to reveal answers to these open questions. If you snooze, you lose those uncomely grayish-brown crescents below your eyes. If you don't snooze, you lose a lot more. The body can't fi ght off infection, the muscles can't regenerate as quickly, the mind can't learn new words, and the eyes can't focus on the road. You also gain things: a bad mood and increased risk for diabetes, high blood pressure, and heart problems. Indeed, the effects of sleep deprivation can be so serious that some sleep scientists liken lifetime sleep debt to a heavy backpack: every sleep hour missed adds an extra pound to your pack until it weighs you down.
For people without time for a daily eight hours in the sack, drugs that counteract the effects of sleep deprivation could serve as substitutes. In a new study, Sam Deadwyler and colleagues have explored this possibility by giving dog-tired rhesus monkeys a drug shown to improve the functioning of alert brains. They found that sleepy monkeys taking the drug performed tasks better and had increased metabolic activity in several regions of their brains. This suggests that the cognitive effects of sleep deprivation can be reduced chemically.
The researchers kept the monkeys awake for 30 to 36 hours by playing music and videos, keeping the lights on, and interacting with them: all the annoyances that can also keep humans from sleeping. To determine the drug's effect on drowsy monkeys, Deadwyler and colleagues used a behavioral test called Match-To-Sample, which measured both accuracy of memory and speed of recall. In the behavioral test, the monkeys saw a simple image fl ash on a screen. For a variable amount of time, the monkeys had to remember the image. Then, they had to select the correct image from a group of others shown on the monitor simultaneously. When monkeys correctly selected the original image with a cursor, they got a squirt of juice in their mouth as reward. The researchers measured how long they could keep the screen blank between the fi rst and second images without affecting the monkeys' performance. They found that if the monkeys were tired, they couldn't remember the fi rst image for long as they could when they were alert. But with the drug, the sleepdeprived monkeys did at least as well as alert monkeys.
The drug, labeled CX717 (Cortex Pharmaceuticals), acts on AMPA receptors, protein structures on the surface of neurons. When these receptors bind to the neurotransmitter glutamate, they transduce excitatory signals by opening an ion channel. Ampakines including CX717 make the activated channel stay open longer when glutamate binds. More ions pass through the channel, creating a stronger signal when nerve cells are activated. The ubiquity of these receptors makes them good targets for drugs that increase general cognitive functioning. The researchers used a technique called positron emission tomography, or PET, to gain insight into CX717's neurobiological role. The PET signal refl ected the distribution and rate of metabolism of ingested radioactively labeled glucose in the monkeys' brain cells. By measuring regional brain glucose metabolism, the researchers determined that for sleepdeprived monkeys, glucose metabolism drops off in brain areas previously associated with memory tasks-namely, the prefrontal cortex, the dorsal striatum, and the medial temporal lobe. However, when sleep-deprived monkeys took the drug, they showed heightened glucose metabolism in these same brain regions. The researchers compared these results to suggest a biological basis for the drug's effects.
Previous studies have shown that caffeine and amphetamine can reduce the deleterious cognitive effects of sleep deprivation. But as anybody who has indulged one latte too many knows, caffeine and other powerful stimulants have limited usefulness. These potentially addictive chemicals can distort thinking just as they can enhance it. Because CX717 has a different biochemical action, it may be more benefi cial than stimulants for counteracting the cognitive effects of sleep deprivation. But that doesn't mean we should throw away our pillows and blankets just yet: sleep deprivation affects both body and mind. Obesity is a leading cause of preventable death and is often linked to type II diabetes and heart disease. Being a complex trait, obesity is likely caused by the interplay of multiple environmental factors and many genes. Common genetic differences between individuals within a region of Chromosome 10 have previously been associated with obesity. This region contains several genes with the potential to be directly involved in the disease. One of these genes, GAD2, has been the subject of many studies. A new study by Michael Swarbrick, Björn Waldenmaier, Christian Vaisse, and their colleagues takes a new look at GAD2 and provides strong evidence that the gene might not be as relevant to obesity as previously thought.
GAD2 encodes a protein (called GAD-65) involved in the production of GABA, a neurotransmitter involved in a variety of brain functions, including appetite stimulation and energy consumption. Studies in mice have shown that increased levels of GABA result in hunger and overeating. In healthy mice, the levels of GAD2, and hence, GABA, are controlled, making sure that the balance between weight gain and loss is maintained. A 2003 study of a French population found that three genetic mutations in and around the GAD2 gene occurred at a high level in individuals with obesity. The 2003 study, conducted by different researchers, A Genetic Link to Obesity: The Numbers Don't Add Up for GAD2 DOI: 10.1371/journal.pbio.0030321 was also published in PLoS Biology. When Swarbrick et al. surveyed German, Caucasian-American, and Canadian populations for this genetic correlation, however, they found no statistically signifi cant link between obesity and any of the mutations.
There are many possible reasons why different studies may show different results: ethnic differences between populations, as well as behavioral and dietary differences, could account for varying results when it comes to studying a trait as complex as obesity. Also, studies that seek to show an association between genetic differences and complex diseases rely heavily on the statistical power of their tests, which depends on the number of subjects involved. Swarbrick et al. have not only studied 2,359 German, 729 US, and 1,137 Canadian subjects, but also conducted a "meta-analysis"-a statistical analysis of a collection of individual studies-of their data and the previously published data from 1,221 French subjects. Metaanalyses help identify patterns from multiple individual studies that may not be visible in any one study alone, and also help rule out chance differences that may be apparent in one single study. In this case, the meta-analysis showed that when the results from French subjects are put together with the results from other ethnic populations, there is no evidence for a link between changes in GAD2 and obesity.
Although GAD2's role in controlling appetite made it an exciting candidate for a link to obesity-related conditions, Swarbrick et al. show that the numbers simply don't add up. The search for serious obesity gene contenders in this region of Chromosome 10 is all set to continue-and attention can now turn to several other potential gene candidates located nearby. No matter how healthy a life one leads, no person has managed to live much longer than a century. Even though the advances of the modern age may have extended the average human life span, it is clear there are genetic limits to longevity. One prominent theory of aging lays the blame on the accumulation of damage done to DNA and proteins by "free radicals," highly reactive molecules produced by the metabolic activity of mitochondria. This damage is expected to reduce gene expression by damaging the DNA in which genes are encoded, and so the theory predicts that the most metabolically active tissues should show the greatest age-related reduction in gene expression. In this issue, Michael Eisen and colleagues show that the human brain follows this pattern. A similar pattern-which, surprisingly, involves different genes-is found in the brain of the aging chimpanzee.
The authors compared results from three separate studies of age-related gene expression, each done on the same type of DNA microarray and each comparing brain regions in young versus old adult humans. In four different regions of the cortex (the brain region responsible for higher functions such as thinking), they found a similar pattern of age-related change, characterized by changes in expression of hundreds of genes. In contrast, expression in one non-cortical region, the cerebellum (whose principal functions include movement), was largely unchanged with age. In addition to confi rming a prediction of the free-radical theory of aging (namely, that the more metabolically active cortex should have a greater reduction in gene activity), this is the fi rst demonstration that age-related gene expression patterns can differ in different cells of a single organism.
The authors found a similar difference in age-related patterns in the brain of the chimpanzee, with many genes down-regulated in the cortex that remained unchanged in the cerebellum. However, the set of affected cortical genes was entirely different between humans and chimps, whose lineages diverged about 5 million years ago. The explanation for this difference is unknown, but the fi nding highlights the fact that signifi cant changes in gene expression patterns, and thus changes in many effects of the aging process, can accumulate over relatively short stretches of evolutionary time.
These results raise a number of questions about age-related gene expression changes, including whether metabolically active non-brain tissues display similar patterns of changes, and whether the divergence between human and chimp patterns was the direct result of selection, or was an inevitable consequence of some other difference in brain evolution. The patterns seen in this study also provide a starting point for understanding the network of genetic changes in aging, and may even reveal targets for treatment of neurodegenerative diseases. Eyewitness testimony has a unique ability to convince juries. The attorney asks the witness to identify the guilty party. The witness points to the defendant, the crowd gasps, and the judge pounds her gavel, demanding order in the court. The jurors casually scribble something in their notes, and everybody knows that the fate of the accused has been sealed. But how reliable is a witness's memory, especially after rehearsing the testimony ad nauseam with a team of lawyers? When a witness presents testimony, is she really remembering the event, or is she remembering something she remembered? Does the initial memory remain intact, or does it degrade like a copy of a copy?
The status of witness testimony in court is just one reason neuroscientists want to understand the biochemical underpinnings of memory formation. Consolidation, the process of new memory formation that takes place in the hippocampus, requires certain proteins. Reconsolidation, the reactivation of these memories in the amygdala, requires a different set of proteins. In the past, neuroscientists hypothesized that reconsolidation might allow old and new memories to link up. A new study by Cristina Alberini and colleagues provides evidence that when rats link new memories to old, the molecular basis of this process actually resembles consolidation.
To manipulate lab rat memories, the researchers used constructions called inhibitory avoidance apparatuses. The fi rst apparatus had two tiny rooms: a well-lit safe room and a pitch-black electric-shock room. Rats spent ten seconds in the fi rst room, the researchers fl ipped on a light, and the rats entered the shock chamber.
Alberini and colleagues knew that the rats had formed a new memory when they hesitated to enter the dark room.
Rats then entered a second apparatus decorated differently from the fi rst apparatus. The safe room smelled of perfume, the walls displayed striped wallpaper, and the fl oor was made from smooth plastic. For rats in the second apparatus, the researchers fl ipped on a light but did not let the rats pass into the shock room. Alberini and colleagues deduced that the rats had compiled their memories of both the fi rst and second apparatuses when they hesitated to enter the second dark room during a fi nal test.
The researchers found that rats injected with anisomycin, a drug that inhibits protein synthesis, could not form a new memory of the second apparatus and sometimes forgot the fi rst. This showed that, as predicted, both the formation of new memories and the reconsolidation of old memories require protein synthesis. The researchers demonstrated the distinction between the processes of consolidation and reconsolidation by showing that rats require a certain protein in the hippocampus only for memory consolidation and the same protein in the amygdala only for reconsolidation.
Using a combination of proteins that took advantage of the differences between consolidation and reconsolidation, the researchers inhibited either the rats' consolidation mechanism or the reconsolidation mechanism.
Then, Alberini and colleagues tested the rats' ability to link their memory of the fi rst apparatus to their exposure to the second. Upon repeated trials, the rats with blocked reconsolidation pathways successfully linked memories of both apparatuses, while the rats with blocked consolidation pathways did not. Therefore, the consolidation pathway, and not the reconsolidation pathway, plays a role in memory linkage.
As a cautionary word, the researchers emphasized that their results applied to the fear-based memories created by the electric shock. Future studies may reveal if other types of memory yield the same results.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Chemistry Biology

text:
Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity

Sulfatide derived from the myelin stimulates a distinct population of CD1d-restricted natural killer T (NKT) cells. Cis-tetracosenoyl sulfatide is one of the immunodominant species in myelin as identified by proliferation, cytokine secretion, and CD1d tetramer staining. The crystal structure of mouse CD1d in complex with cis-tetracosenoyl sulfatide at 1.9 Å resolution reveals that the longer cis-tetracosenoyl fatty acid chain fully occupies the A′ pocket of the CD1d binding groove, whereas the sphingosine chain fills up the F′ pocket. A precise hydrogen bond network in the center of the binding groove orients and positions the ceramide backbone for insertion of the lipid tails in their respective pockets. The 3′-sulfated galactose headgroup is highly exposed for presentation to the T cell receptor and projects up and away from the binding pocket due to its β linkage, compared with the more intimate binding of the α-glactosyl ceramide headgroup to CD1d. These structure and binding data on sulfatide presentation by CD1d have important implications for the design of therapeutics that target T cells reactive for myelin glycolipids in autoimmune diseases of the central nervous system.

Innate pathways of immunity are important not only in providing a rapid response against infectious agents, but also in the effective induction and/or modulation of adaptive responses. Natural killer T (NKT) cells that share the cellsurface receptors of NK cells and, in addition, express an antigen receptor (TCR) are generally stimulated by lipid antigens and rapidly secrete large amounts of IFN-␥ and IL-4 after activation (1). NKT cells influence the outcome of the immune response against tumors and infectious organisms, as well as the course of several autoimmune diseases (2,3).
NKT cells recognize lipid antigens when bound by nonpolymorphic, MHC class I-like CD1 molecules (4). APCs, such as dendritic cells, macrophages, and subsets of B cells, express CD1 molecules. The CD1 family consists of five distinct genes (CD1a-e) that can be categorized into two groups. In humans, group 1 consists of CD1a, CD1b, CD1c, and CD1e, whereas CD1d is the only member of group two and is also expressed in mice.
The first crystal structure of mouse CD1d revealed a deep hydrophobic ligand binding groove, which seems to be well-suited for the binding of long chain hydrophobic compounds, such as lipids, where electron density for two long aliphatic chains was observed, but not fully interpreted, in the binding groove (5). Recently, this bound self-ligand was identified as phosphatidyl choline (PC), and the CD1d crystal structure confirmed that the two lipid tails are inserted into the A Ј and F Ј pockets, with the headgroup extending out of the groove for T cell recognition (6).
Because different lipids are either present in or traffic to distinct endocytic compartments, it has been suggested that the different CD1 isotypes have evolved to target these various intracellular locations. CD1b and CD1d are localized mainly in the late endosomes or lysosomes where microbial lipids accumulate after infection, whereas CD1a recycles back to the cell surface via passage through early or recycling endosomes (14,15). Recently, a family of endosomal lipid transfer proteins, the saposins, has been shown to be necessary for the presentation of certain lipids by CD1. It has been proposed that these saposins have the ability to extract and bind monomeric lipids from the membrane, which could then be transferred through a yet unknown mechanism to CD1 in the endosomal compartments (16)(17)(18).
Characterization of lipid-reactive NKT cells and their function is important for understanding the role of this NKT cell subset in the spectrum of immune responses. NKT cells have regulatory effects in both experimental models and in human autoimmune diseases, such as diabetes and multiple sclerosis (2,3). These cells are autoreactive and express a memory/activation phenotype. NKT cells are heterogeneous, but most of their phenotypic and functional characteristics have been derived from studies of the major mouse NKT cell population that expresses TCRs with an invariant V ␣ 14 ϩ ␣ -chain (V ␣ 24 ϩ in human). A nonmammalian glycolipid, ␣ -GalCer, has been invaluable as a highly potent, stimulatory ligand for these NKT cells in the context of CD1d. ␣ -GalCer, unlike most mammalian glycolipids, has its carbohydrate moiety attached via an ␣ rather than a ␤ linkage. Thus, very little is known about the phenotype or physiological relevance of self-glycolipid-reactive NKT cells compared with the ␣ -GalCer-reactive invariant NKT cells.
In an attempt to characterize self-glycolipid recognition by NKT cells and, more precisely, to elucidate the structural differences in ligand binding and NKT cell stimulation between ␣ -anomeric ( ␣ -GalCer) versus ␤ -anomeric (sulfatide) glycolipids by mouse CD1d, we crystallized CD1d in complex with a synthetic sulfatide self-antigen and determined its three-dimensional structure at 1.9 Å resolution.

Sulfatide recognition by CD1-restricted T cells
One of the most abundant galactolipids in the axon-insulating myelin sheet of the CNS is 3 Ј sulfogalactosyl ceramide (sulfatide), in which the 3 Ј OH of galactose is sulfated and the sugar is attached to the ceramide via a ␤ -glycosidic link- (A) Dominant response to cis-tetracosenoyl sulfatide as examined by in vitro proliferation to a titrated dose of cis-tetracosenoyl (C 24:1 ) sulfatide (᭡), tetracosanoyl (C 24:0 ) sulfatide (᭝), lyso-sulfatide (no fatty acid; ᭹), and palmitoyl (C 16:0 ) sulfatide (᭺) in splenocytes from C57BL/6 mice. One of three representative experiments is shown. (B) Loading of cis-tetracosenoyl sulfatide onto mouse CD1d molecules. 9 g of purified mouse CD1d protein was incubated at 37ЊC for 6 h with 4 g of purified sulfatide (sulf) from bovine brain or synthetic cis-tetracosenoyl sulfatide (cis) and subjected to IEF gel electrophoresis. One of two representative experiments is shown. (C) Staining of mononuclear cells from liver with cis-tetracosenoyl sulfatide CD1d/tetramer. Flow cytometric analysis of liver mononuclear cells from CD1d ϩ/ϩ (C57BL/6, panels 1 and 2, or SJL/J, panel 3) and C57BL/6.CD1d Ϫ/Ϫ mice (panel 4) after staining with either cis-tetracosenoyl sulfatide loaded or unloaded CD1d tetramers, which were labeled with phycoerythrin (PE) and anti-TCR-␤-FITC. In each panel, the numbers indicate the percent of positive cells. One of three representative experiments is shown. (D) Proliferation and cytokine secretion response of splenocytes from naive C57BL/6 () or C57BL/6.CD1d Ϫ/Ϫ (ٗ) mice to an in vitro stimulation with a titrated dose of cis-tetracosenoyl sulfatide. One of five representative experiments is shown.

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age. We have shown that sulfatide is recognized by a subset of CD1d-restricted T cells that are distinct from the invariant V ␣ 14 ϩ T cells that respond to the potent glycolipid, ␣ -Gal-Cer, as well as to a sphingolipid self-antigen, iGB3, and the microbial glycuronosyl ceramides (4,20). Sulfatides derived from the brain are comprised of several molecular species that differ in length of their acyl chain and in the extent of the unsaturation and hydroxylation of the N-amide-linked fatty acid of the ceramide backbone. Thus, natural sulfatide is a 2:1 mixture of saturated and unsaturated fatty acyl chains with tetracosanoyl (C 24 ) constituting the predominant chain length (22).
To analyze the fine specificity of sulfatide-reactive T cells, we have used synthetic homologues or analogs of sulfatide comprised of different fatty acid chain lengths (C 16 -C 24 ) to characterize the immunodominant sulfatide species. In vitro proliferation or cytokine secretion assays were used to determined the most active species as cis-tetracosenoyl (C 24:1 , mono-unsaturated fatty acid) sulfatide ( Fig. 1 A). Other sulfatides, including tetracosanoyl-(C 24 , saturated fatty acid), palmitoyl-(C 16 , saturated fatty acid), and lysosulfatides (lacking the fatty acid chain) did not induce a significant immune response ( Fig. 1 A). Furthermore, adjuvantfree administration of cis-tetracosenoyl sulfatide in three different mouse strains resulted in significant amelioration of a chronic and relapsing form of EAE (unpublished data). Sulfatides, either naturally derived from bovine brain or as synthetic cis-tetracosenoyl species, can be efficiently loaded onto purified mCD1d, as judged by isoelectric focusing gel electrophoresis ( Fig. 1 B). The negative charge on the sulfatide promotes a shift in the migration of exogenously loaded mCD1d compared with endogenous CD1d, which is likely loaded with a neutral self-antigen, such as PC (6).
Next, we determined whether synthetic cis-tetracosenoyl sulfatide is able to form mCD1d-tetramers that can stain NKT cells, similar to the recent experiments with tetramers that were loaded with natural sulfatides from bovine brain (19). In two different CD1d ϩ / ϩ mouse strains, around 1-2% of T cells can be stained with cis-tetracosenoyl/mCD1d-tetramers, whereas no staining was observed in CD1d Ϫ / Ϫ mice or when unloaded tetramers were used to stain NKT cells in a CD1d ϩ / ϩ mouse ( Fig. 1 C). To examine the nature of immune response to cis-tetracosenoyl sulfatide and its dependence on the CD1d antigen presentation pathway, spleen cells from naive wildtype mice, as well as CD1d Ϫ / Ϫ mice, were cultured with different concentrations of the synthetic glycolipid and assayed for proliferation as well as IFN-␥ and IL-4 secretion (Fig. 1  D). Splenocytes from wild-type mice, but not from CD1d Ϫ / Ϫ mice, proliferated and produced IFN-␥ in response to cis-tetracosenoyl sulfatide. No significant secretion of IL-4 was observed under these experimental conditions.
The sulfatide negative charge arises from the 3 Ј sulfate group. Because mono-GM1 that contains sialic acid, which is also negatively charged, does not induce proliferative/cytokine responses, and because GM1-CD1d-tetramers do not stain NKT cells (unpublished data), it seems unlikely that the negative charge alone is responsible for specific recognition by NKT cells. In addition, the 3 Ј sulfate group is required for the activation of sulfatide-specific NKT cells because ␤ -GalCer, which is identical to the sulfatide but lacks the 3 Ј sulfate group, does not stimulate this NKT cell pool. As 3 Јsulfated ␣ -GalCer is also not able to activate sulfatide-specific NKT cells (unpublished data), it is suggested that these sulfatide-reactive NKT cells are specific for the configuration of the sulfated galactose moiety ( ␤ form active, ␣ form inactive) at the CD1d surface.

Structure determination of the CD1d-sulfatide complex
Soluble and fully glycosylated, heterodimeric mouse CD1d-␤ 2 M protein (residues 1-279 heavy chain and 1-99 ␤ 2 M) was secreted by Spodoptera frugiperda (SF9) cells upon infection with recombinant baculovirus and purified to homogeneity by affinity and size-exclusion chromatography, as described below in Materials and methods. After partial deglycosylation of the protein using Endoglycosidase H (Endo H), synthetic cis-tetracosenoyl sulfatide was loaded by incubating CD1d with a 10-fold molar excess of sulfatide without any detergents. The CD1d-sulfatide complex was further purified by size-exclusion chromatography. More than 95% of the CD1d protein appeared to be loaded with sulfatide, as judged by native IEF gel electrophoresis (unpublished data). Using the sitting drop vapor diffusion method, we crystallized the CD1d-sulfatide complex and determined its three-dimensional structure by molecular replacement using the protein coordinates of the CD1d short-chain ␣-Gal-Cer complex (PDB code 1Z5L) as the search model (Table I and Fig. 2; reference 11). The crystal structure was refined to a final R cryst and R free of 18.6 and 24.8%, respectively. The asymmetric unit of the crystal contains two CD1d-sulfatide complexes (A and B), which are very similar (root-meansquare deviation of 0.55 Å for all C ␣ atoms). Thus, we describe here only the crystal structure for complex A, except where stated otherwise.

Structural features of the mouse CD1d-sulfatide complex
The overall architecture of mouse CD1d has been described (5,6,11). The binding groove of CD1d is formed by two anti-parallel ␣-helices that sit atop a six-stranded, ␤-sheet platform and is lined mainly by hydrophobic residues (Fig. 2 A). The two major pockets, AЈ and FЈ, are located deep inside the CD1 protein and each accommodate one alkyl chain of the sulfatide ligand. The sulfated galactose headgroup is then positioned above the binding groove at the interface between both pockets (Fig. 2 B). As the AЈ pocket is larger in volume than the FЈ pocket, the synthetic sulfatide ( Fig. 2 C, left) binds such that the longer cis-tetracosenoyl fatty acid (C 24:1 ) occupies the AЈ pocket, whereas the shorter sphingosine backbone (C 18 ) is inserted into the FЈ pocket. As the sulfatide alkyl chains then fully occupy both binding pockets, the previously reported spacer lipid (11) is not observed in the CD1d binding groove. Although the electron density is relatively weak at the junction where the cis-tetracosenoyl fatty acid starts to encircle the central pole of the AЈ pocket, the fatty acid appears to go around the AЈ pole in a counterclockwise direction when looking down into the binding groove (Fig. 3, A and B). This conformation places the cisunsaturation in the middle of the fatty acid curvature, which could facilitate the encircling of the pole. Several water molecules are located in the vicinity of the polar groups of the sulfatide, mainly proximal to the galactosylsulfate headgroup, which suggests that these waters play a role in the CD1ligand interaction (Figs. 3 and 4). In contrast, no water mol-ecules could be observed at similar locations in the CD1d short-chain ␣-GalCer (PBS-25) structure (11), likely due to the closer binding of the ␣-linked galactose to the ␣2-helix of CD1d. As in the CD1d-PBS-25 structure, the electron density is best defined for the polar region of the sphingosine backbone and the connection with the fatty acid (Fig. 3), as hydrogen bonds with CD1d residues stabilize and provide specificity for this region of the ligand (Fig. 4). Seven hydrogen bonds are formed directly between CD1 and the sulfatide, whereas two additional hydrogen bonds are mediated by water molecules-one between the sulfate group and the backbone nitrogen of Gln154 and the other between the keto group of the fatty acid and the backbone oxygen of Met69. The same CD1 residues are involved in hydrogen bond formation with the short-chain ␣-GalCer ligand PBS-25. Although several residues of the ␣1-helix (Asp80, Arg79, and Met69) provide specific contacts to the ceramide back- The sulfatide fatty acid is bound in the larger AЈ pocket, and the sphingosine backbone is bound in the FЈ pocket. N20, N42, and N165 represent the three ordered N-linked glycosylation sites that carry at least the remaining proximal N-acetylglucosamine residue after deglycosylation. (C) The chemical structure of the sulfatide used in this study and other CD1d ligands, such as PC (6), iGB3 (20), and the short-chain ␣-GalCer variant PBS-25 (11) for structural comparison. The length of the individual alkyl chains is given as the number of carbon atoms (C 12 -C 26 ).

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bone, the ␣2-helix (Asp153, Thr156, and Gln154) is more involved in stabilizing the galactosyl headgroup. Nevertheless, Arg79 hydrogen bonds to the 6Ј hydroxyl group of the galactose, thereby fixing the orientation of galactose between the ␣1and ␣2-helices in concert with Asp153, which interacts with the 2Ј hydroxyl group on the opposite side of the galactose. The sulfate group is only loosely bound, which explains its high B-values (07فÅ 2 ) compared with the rest of the ligand (04فÅ 2 ) or the surrounding CD1 residues (24-53Å 2 ).
Although the sulfated galactose headgroup makes an additional hydrogen bond with CD1d, as compared with the short-chain ␣-GalCer variant PBS-25 (11), its electron density is less well-defined. The ␤-linkage between the galactose and the ceramide backbone in the sulfatide results in a much higher degree of solvent exposure of the headgroup, whereas the two short hydrogen bonds (2.5Å and 2.6Å) between Asp153 and the ␣-linked galactose of PBS-25 pull the galactose toward the ␣2-helix in an orientation horizontal to the CD1 surface, which results in more intimate contacts with CD1d, as reflected by similar B-values of the PBS-25 galactose and the surrounding CD1 residues.
Comparison of ␣versus ␤-anomeric ligands A comparison of the orientation of the sulfatide and the PBS-25 ligand in the mCD1d binding groove (Fig. 5, A and B) illustrates that their lipid backbones are bound in a similar location, likely due to the specific hydrogen bonding between the ceramide moiety and the CD1 residues, whereas the different linkage between the galactose headgroups and the lipid (␣ vs. ␤) results in conformational differences between each CD1d-lipid complex. The ␤-anomeric galactose (sulfatide) is highly exposed and projects out of the binding groove perpendicular to the ␣1and ␣2-helices (Fig. 5 A), whereas the  ␣-anomeric galactose (PBS-25) sits flat atop the binding groove between both helices, thereby burying the lipid backbone while simultaneously exposing less surface area for recognition by the semi-invariant V␣14 NKT cell receptor (Fig.  5 A and B, bottom). However, the short-chain ␣-GalCer ligand PBS-25 induces the formation of a roof above the FЈ pocket (Fig. 5 B) from CD1d residues, which can now play a role in CD1d-TCR interaction. Small chemical differences in the structure and binding of CD1d-specific ligands can then be directly transmitted to the CD1d surface and, therefore, can aid in ligand discrimination by NKT cells. In the case of the sulfatide ligand, where the galactosylsulfate moiety sticks out of the groove, interaction with the variant sulfatide-reactive NKT cells is likely to be different from iNKT cells. Here, the TCR has to accommodate a more protruding headgroup and a different conformation of residues on the CD1d surface. In addition, the sulfatide ligand exposes more of its lipid backbone, which could now play a role in the T cell-recognition process and help explain the biological differences among various sulfatide species that have different length fatty acid chains (Fig. 1 A).

Comparison of sulfatide binding by CD1d and CD1a
To compare the binding of sulfatides to different CD1 isotypes, the mouse CD1d-sulfatide (Fig. 5 A) complex was analyzed against the previously determined CD1a-sulfatide structure (Fig. 5 C; reference 7). In both cases, the ␤-linked galactose moieties face upward and are either sandwiched between both ␣-helices (CD1a) or 90Њ rotated, sitting above the binding groove (mCD1d). In each case, the sulfate is the most exposed group of the ligand and the major antigenic epitope, as sulfate-lacking ␤-GalCer is not antigenic (unpublished data). Interestingly, the sulfate ligand is inserted into the two CD1 binding grooves in opposite orientations. In CD1a, the sphingosine backbone occupies the fixed-size AЈ pocket (C 18 , molecular ruler), and the fatty acid tail end ex-tends out of the FЈ pocket (C 18 ) at the CD1 surface. In mCD1d, the AЈ pocket is much larger and fully accommodates the longer fatty acid chain (C 24 ), whereas the sphingosine backbone now occupies the smaller FЈ pocket. In either case, the more restricted of the two CD1 binding pockets binds the fixed-length sphingosine chain, whereas the larger (CD1d) or more open-ended (CD1a) pockets bind the more variable fatty acid. Thus, two different modes of sulfatide binding are found in these CD1 isoforms. In CD1a, the binding groove is fully occupied by the ligand, and any variation in the length of the fatty acid can be accommodated by extension outside the binding groove, which can then directly translate into possible direct contact with the TCR. In mCD1d, the shorter fatty acid chains result in partial filling of the AЈ pocket. Depending on the extent of the AЈ pocket that is not occupied by a ligand, a pocket-stabilizing factor, as observed for the short-chain ␣-GalCer variant PBS-25, is then recruited to fill up the unoccupied cavity to form a stable complex (11). In mouse CD1d, the corresponding T cell would not directly see variations in fatty acid chain length at the CD1 surface, whereas for CD1a, these subtle differences in length can directly affect T cell stimulation (23).

DISCUSSION
The CD1 family of glycosylated cell-surface receptors constantly surveys the lipid content of APCs by intracellular trafficking through the various endosomal compartments. The number and different classes of lipids that are now known to act as ligands at the cell surface of APCs for specific T cells have increased significantly in the last few years and include phosphoglycerolipids, sphingolipids, diacylated glycerolipids, lipopeptides, mycolates, phosphomycoketides, and small hydrophobic compounds (for review see references [13][14][15]. Some of these CD1 ligands promiscuously bind to all CD1 isotypes (e.g., sulfatide), whereas others are isoform-specific. For example, mycolates are only bound to CD1b, whereas . Although the galactose of sulfatide presented by CD1d is more exposed and sticks out from the center of the groove, that of PBS-25 sits more closely above the CD1d surface while burying the lipid backbone underneath. (C) The stearoyl sulfatide (C 18:0 ) ligand is more nestled in the CD1a-binding groove (PDB code 1ONQ), whereas the fatty acid tail end is exposed at the CD1 surface.

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phosphomycoketides bind to CD1c. These observations were at first surprising. Sulfatides and lipopeptides, which have quite different chemical composition, can both bind to CD1a but do so in a way that does not alter the structure of the CD1a-binding groove. Both ligands maneuver into the binding groove and attain an optimal fit without inducing any changes in the CD1 conformation (7,8). In contrast, structures of mCD1d in complex with either PC, sulfatide, or the short-chain ␣-GalCer PBS-25 show interesting structural changes at the T cell-recognition surface of CD1. Compared with the first crystal structure of mouse CD1d, to which an endogenous PC ligand was bound (5,6), binding of the short-chain ␣-GalCer variant results in an alteration at the CD1 surface, which leads to the formation of a roof above the FЈ portal (11). This induced fit, however, has not been observed in the structure of mCD1d-sulfatide. Although both sulfatide and ␣-GalCer are glycosphingolipids, they differ in their chemical structure, not only in the linkage to their galactosyl headgroups, but also in their ceramide lipid backbone. ␣-GalCer is based on phytoceramide, which has 3Ј and 4Ј OH groups, whereas the ceramide of sulfatide has a 4Ј OH group and a 4Ј-5Ј unsaturation. This additional 4Ј OH group of ␣-GalCer forms a second hydrogen bond with Asp80, and the ␣-anomeric galactose forms tight hydrogen bonds with Asp153 of the ␣2-helix. We propose that this tighter hydrogen bond interaction of ␣-GalCer with both ␣-helices results in the structural changes observed in the mouse CD1d-␣-GalCer complex. PC is a glycerolipid and its lipid backbone diacylglycerol, as well as the phosphorylcholine headgroup, interestingly form fewer hydrogen bonds with CD1 and, therefore, do not appear to induce any structural changes, although no truly "empty" structure is known. However, the crystal structure of human CD1d with bound full-length ␣-GalCer shows that under certain experimental conditions, such as refolding without ligands, the protein can undergo structural changes that partially close the binding groove. Two CD1d molecules were observed in the crystal structure: one was loaded with ␣-GalCer, whereas the other was empty. However, whether this capacity of human CD1d to close the binding groove in absence of any detectable ligand is biologically significant remains elusive. Under physiological conditions, such as during folding in the ER, lipids are always present to stabilize the hydrophobic CD1d binding groove, thereby rendering any requirement for gross structural changes unnecessary.
All of the four lipid ligands that have been crystallized in complex with either human (12) or mouse (6, 11) CD1d so far show a common mode of lipid binding. The C 18 sphingosine chain is always inserted in the FЈ pocket, and fatty acid (C 8 -C 26 ) is always inserted in the AЈ pocket. This orientation is similar for PC, where the shorter fatty acid chain (C 12 ) is inserted in the AЈ pocket and the longer fatty acid (C 24 ) in the FЈ pocket. Nevertheless, a sphingolipid with a C 18 fatty acid, as found in sulfatides from natural sources, could insert its two alkyl chains in either pocket if the length of the alkyl chain is the only factor responsible for ligand binding. But the crystal structure of the CD1d-␣-GalCer (PBS-25) complex revealed that the short fatty acid (C 8 ) is surprisingly inserted only in the AЈ pocket. The orientation still needs to be determined for the recently identified family of ␣-anomeric microbial ligands, the glycuronosyl ceramides (C 14 ; references 24 and 25). Thus, we propose that the N-amide linkage between the fatty acid and the sphingosine together with the hydroxyl groups of the sphingosine chain (Fig. 2 C, highlighted in yellow), which forms a precise hydrogen-bonding network with CD1, constrains the ceramide backbone that orients the respective tails into the AЈ and FЈ pockets.
It is not known whether the immunodominance of cistetracosenoyl sulfatide is due to a more efficient binding to CD1d or to the presence of specific TCR repertoires that are stimulated preferentially by individual sulfatide species. In this regard, it is interesting to note that the T cell hybridoma specific for lyso-sulfatide does not recognize any of the other sulfatides examined so far (unpublished data). Whether NKT cells specific for each individual sulfatide express a unique TCR or whether overlapping TCR repertoires exist is currently not clear and should be determined by generating T cell hybridomas reactive to each of the individual sulfatides. Of interest, however, is that although the hydrogen bonding network formed between the lipid backbone and CD1d is responsible for the orientation of the different lipid ligands in the binding groove, slight differences in the positioning of sulfatide and ␣-GalCer can be observed when both structures are superimposed (not depicted). The same is true when the binding of ␣-GalCer to CD1d is compared in both mouse and human crystal structures. The exchange of one residue at the CD1d surface (Trp153 in human and Gly155 in mouse) results in a different positioning of the galactose, whereas the lipid backbone is bound in the same orientation (26). Therefore, it is possible that the biological differences between the different sulfatide species shown in Fig.  1 A are not only the result of differential loading characteristics of the ligands, but rather the consequence of subtle structural differences upon ligand binding due to either differences in fatty acid chain length and/or saturation (C 16 vs. C 24:1 ), or, in case of lyso-sulfatide, the complete lack of a fatty acid chain. However, it cannot be completely ruled out that the stronger NKT cell activation by cis-tetracosenoyl sulfatide is a result of the increased solubility of the monounsaturated and more polar nervonyl (C 24:1 ) fatty acid versus the fully saturated tetracosanoyl (C 24:0 ) fatty acid, which could lead to an increased efficiency of loading onto CD1.
Recognition of sulfatide by CD1d-restricted NKT cells has important implications in autoimmune diseases of the CNS, as sulfatides are one of the major glycolipid components of the myelin membranes that are targeted in such diseases as multiple sclerosis (MS). MS is a demyelinating disease mediated by a T cell-guided immune response that is either initiated from antigen-presenting events in the CNS or induced after the peripheral activation by a systemic molecular mimicry response (27,28). Indeed, in MS patients, increased serum levels of glycolipids (29,30) and antibodies directed against them have been reported (31)(32)(33)(34). T cells specific for glycolipids have been isolated from MS patients. Their frequency in five active MS patients was three times higher compared with five normal individuals (35). Recently, it has been demonstrated that sulfatide binds promiscuously to all of the CD1a, CD1b, CD1c, and CD1d isoforms (36). Because CD1 molecules are up-regulated on macrophages in areas of demyelination in chronic-active MS lesions but not in silent lesions in the brain (37), it is possible that self-glycolipids from myelin could be presented during local inflammation to T cells. Microglia, as well as infiltrating macrophages, could either engulf myelin components or internalize them by Fc receptors or by complement receptor-mediated phagocytosis after binding to myelin-specific antibodies. Thus, activated APCs in the CNS could not only present peptides (MHC) to T cells, but also glycolipids (CD1). Thus, myelin glycolipid-reactive T cells could potentially influence the inflammation and demyelination in the CNS. It is clear from our data (19 and unpublished data) that the peripheral activation of sulfatide-reactive T cells after adjuvant-free administration of sulfatide ameliorates experimental autoimmune disease of the CNS. Because CD1 molecules, unlike the classical MHC molecules, are nonpolymorphic, insight into the molecular recognition of sulfatide by the CD1d molecules and their interaction with a unique CD1d-restricted NKT cell population will be extremely valuable in the potential development of non-HLAdependent therapeutic approaches for autoimmune demyelinating diseases in humans.

MATERIALS AND METHODS
Mice. 6-8-week-old female C57BL/6 or SJL/J (CD1d ϩ/ϩ ) mice were purchased from The Jackson Laboratory. C57BL/6 (CD1d Ϫ/Ϫ ) mice were originally generated in the laboratory of L. Van Kaer (Vanderbilt University, Nashville, TN) and were provided by M. Kronenberg (La Jolla Institute of Allergy and Immunology, La Jolla, CA). All animals were housed under specific pathogen-free conditions, and animal protocols were approved by the Institutional Animal Care and Use Committee of the Torrey Pines Institute for Molecular Studies.
Cell preparation. Hepatic lymphocytes were isolated using established Percoll gradient methods. In brief, animals were killed by CO 2 inhalation and perfused with chilled PBS. To obtain lymphocytes, the liver was removed, cut into small pieces, passed through a 70-m nylon cell strainer (Falcon; Becton Dickinson), and suspended in DMEM supplemented with 2% heat-inactivated FBS. After centrifugation (1,500 g for 10 min), the cell pellet was washed and resuspended in the same medium. Lymphocytes were isolated from parenchymal hepatocytes, nuclei, and Kupffer cells by Percoll (35% Percoll containing 100 U/ml heparin) gradient separation. Splenocytes were obtained by passing the spleen through a 70-m nylon strainer and suspension in DMEM with 2% heat-inactivated FBS. Erythrocytes were lysed with RBC lysis buffer (BD Biosciences).
Monoclonal antibodies and flow cytometry. Mononuclear cells were isolated and suspended in FACS buffer (1-2 ϫ 10 6 /ml) containing 0.02% NaN 3 in PBS (wt/vol) and 2% FBS (vol/vol) and treated with antibodies against FcR-␥ (2.4G2) to block nonspecific binding and then labeled with the indicated monoclonal antibodies. For flow cytometry staining, 2.5 g of tetramerized mCD1d was used. Flow cytometric analysis was performed on a FACSCalibur instrument using CELLQuest software (Becton Dickinson).
Tetrameric mCD1d-lipid complexes. Mouse CD1d protein was generated using the baculovirus expression system essentially as described previously (19). Unloaded mCD1d tetramers were prepared by preincubating biotinylated mCD1d protein with vehicle only. To produce glycolipid-mCD1d tetramers, biotinylated mCD1d protein was incubated with sulfatide at a molar ratio of 1:6 for 18-20 h at 22ЊC.
In vitro loading of sulfatides onto mouse CD1d. Soluble mouse CD1d (9 g/18 l in PBS) was mixed with 4 g (2.0 l) of purified bovine brain-derived sulfatide or the synthetic cis-tetracosenoyl sulfatide and incubated at 37ЊC for 6 h followed by washing with 500 l of PBS and concentrated using 10 kD mol wt cut-off centrifugal concentrators (Millipore). After adjusting the remaining volume, equal amounts of protein were subjected to IEF gel electrophoresis to monitor the loading efficiency of sulfatide onto CD1d.
Protein expression, purification, and crystallization. The baculovirus transfer vector pAcUW51 (BD Biosciences) containing the nucleic acid sequence for the extracellular domains of mCD1d and ␤ 2 M, including signal sequence, was provided by M. Kronenberg. To engineer a CD1d construct for crystallization purposes, the biotinylation domain, generally used for the preparation of CD1 tetramers followed by a hexahistidine tag, was removed by digestion with the restriction enzyme BamHI and replaced with a short hexahistidine tag that was introduced by ligation of two complementary BamHI-restricted oligonucleotides. A SalI site was incorporated in the oligonucleotide to verify the integration of the fragment by restriction digestion. Three colonies were analyzed by sequencing to find one clone with the right orientation of the hexahistidine tag. The constructed plasmid was cotransfected into SF9 cells with BaculoGold DNA using Cellfectin reagent (Invitrogen) according to the manufacturer's specifications. After several rounds of virus amplification, the protein was expressed for 3 d in shaking flasks (145 rpm) at 28ЊC using SF9 cells (2 ϫ 10 6 cells/ml) with a multiplicity of infection of .3ف The expression experiment was typically performed on a 5-8-liter scale. SF9 cells were removed from the cell culture media by centrifugation, and the media was further concentrated to 1 liter and washed several times with PBS buffer. The protein was purified from the concentrated media by ion metal affinity chromatography on a Ni-NTA resin (QIAGEN) in 50 mM Tris/HCl buffer, pH 8.0, followed by anion-exchange chromatography using MonoQ HR 10/10 (GE Healthcare) in 10 mM Tris/HCl buffer, pH 8.0, with a linear gradient of 0-250 ARTICLE mM NaCl. Two of the four total N-linked carbohydrates were removed after the proximal N-acetyl glucosamine residue by Endo H cleavage, as judged by mass spectrometry analysis (unpublished data). In brief, 1 mg CD1d protein was incubated with 50 mU Endo H in 100 mM sodium acetate, pH 5.9, at 37ЊC for 2 h and purified by size-exclusion chromatography (Superdex S200 10/300 GL; GE Healthcare). The synthetic sulfatide ligand was then loaded onto the partially deglycosylated protein by incubating 3 mg CD1d with a 10-fold molar excess of synthetic sulfatide (0.12 ml of a 5 mg/ml solution in DMSO) in 100 mM sodium acetate buffer, pH 5.9, for 6 h at 37ЊC with intermittent agitation. The loaded CD1d protein was purified by ion-exchange chromatography on a MonoQ column using a shallow gradient of NaCl (0-150 mM over 25 column volumes). Loading of sulfatide onto CD1d was observed by native IEF gels that monitored a shift toward the cathode due to the additional negative charge from the sulfate moiety. The CD1d fractions from the MonoQ run were pooled and concentrated to 7.5 mg/ml in 10 mM Hepes, pH 7.5, and 25 mM NaCl. The best crystals were obtained at 22ЊC by mixing 1 l protein (7.5 mg/ml) with 1 l precipitant solution (2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, 0.2 M NaCl, and 10 mM manganese chloride) and grown for 2 wk for data collection.
Structure determination. Crystals were flash-cooled at a temperature of 100 K in mother liquor containing 25% glycerol. Diffraction data from a single crystal was collected at Beamline 8.2.2 of the Advanced Light Source, Berkeley, CA, and then later from another crystal at Beamline 11.1 of the Stanford Synchrotron Radiation Laboratory and processed to 2.7 Å and 1.9 Å, respectively, with the Denzo-Scalepack suite (40) in spacegroup P2 (unit cell dimensions: a ϭ 58.86 Å, b ϭ 74.84 Å, c ϭ 101.56 Å, and ␤ ϭ 102.1Њ). Two CD1-lipid complexes occupy the asymmetric unit with an estimated solvent content of 53.8% based on a Matthews' coefficient (V m ) of 2.66 A 3 /Da. Molecular replacement in P2 1 was performed with the lower resolution dataset in CCP4 (41) using the program MOLREP (42) and the CD1d-PBS-25 structure (15ZL) as the search model, with the ligand removed, which resulted in a R cryst of 48.2% and a correlation coefficient of 0.46. Subsequent rigid-body refinement in REFMAC 5.2 to a resolution of 3.5 Å resulted in an R cryst of 41.4%. The initial refinement included several rounds of restrained refinement against the maximum likelihood target in REFMAC 5.2. Tight restraints were maintained between the two molecules in the asymmetric unit throughout the refinement of the lower resolution dataset. At a later stage of refinement, carbohydrates were built at 5 out of the 8 N-linked glycosylation sites in both CD1 molecules. When the 1.9 Å high resolution dataset became available, it was then used for subsequent refinement of the model derived from the 2.7 Å resolution data. Before model building and restrained refinement, one round of rigid-body refinement was performed to 3.5 Å resolution that resulted in an R cryst of 28%. Tight noncrystallographic symmetry restraints were applied only during the first round of refinement and then not used further, as they resulted in an increase in the R free value. The refinement progress was judged by monitoring the R free for cross-validation (43). The model was rebuilt into A -weighted 2Fo-Fc and Fo-Fc difference electron density maps using the program O (44). Water molecules were assigned during refinement in REFMAC using the water ARP module for Ͼ3 peaks in an Fo-Fc map and retained if they satisfied hydrogen-bonding criteria and returned 2Fo-Fc density Ͼ1 after refinement. Starting coordinates for the sulfatide ligand were obtained from the CD1a-sulfatide structure (1ONQ) and modified accordingly using the molecular modeling system INSIGHT II (Accelrys, Inc.). The sulfatide library for REFMAC (45) was created using the Dundee PRODRG2 server (46). Final refinement steps were performed using the translation, libration, and screw-rotation displacement procedure in REFMAC (47) with a total of six anisotropic domains (three per complex: ␣1-␣2 domain, ␣3-domain, and ␤ 2 M) and resulted in improved electron density maps for the glycolipid ligand and a further drop in R free . The CD1d-sulfatide structure has a final R cryst of 18.6% and an R free of 24.8%. The quality of the model (Table I)


extfieldsofstudy: Chemistry Medicine

text:
Substrate Dependence in Aqueous Diels-Alder Reactions of Cyclohexadiene Derivatives with 1,4-Benzoquinone

A reactivity difference based on the position of substituents on cyclohexa-1,3-diene was observed for the title reaction. The effect of water as solvent was more distinct for 1-methyl-4-isopropylcyclohexa-1,3-diene than for 2-methyl-5-isopropylcyclohexa-1,3-diene or non-substituted cyclohexa-1,3-diene. The effect of NaCl (salting-out) and guanidium chloride (salting-in) was also large for 1-methyl-4-isopropylcyclohexa-1,3-diene.

Introduction
Recently, the development of organic reactions in aqueous media has been remarkable [1], especially in light of the interest in "green chemistry". Among the various reactions, the Diels-Alder reaction is well documented [2,3]. The effect of micelles [4] or Lewis acids [5], including Lewis acid-surfactant combinations [6] have also been studied for the aqueous Diels-Alder reaction. In contrast to the reaction in organic solvents, the water-based reaction proceeds with higher endo/exo selectivities [7]. On the other hand, substrate specificity is one of the fundamental features of enzyme or other biochemical reactions which takes place in water. In such reactions, a simple alkyl substituent sometimes plays an important role. We have studied the substrate dependence of the aqueous Diels-Alder reaction, and here report that the position of the substituents is an important factor in the aqueous Diels-Alder reaction of cyclohexadiene derivatives.
When the reaction of 1 and 4 was carried out in water, the adduct was separated as a precipitate, which was collected after 2 days of stirring at room temperature to afford 5 [8] in 67% yield (Table 1; Entry 1). The dienes having alkyl substituents, 2 and 3, were also treated under the same reaction conditions giving 6 [9] and 7 [10], respectively (Entries 2,3). In order to clarify the effect of the aqueous media, the reaction in organic solvents such as toluene and THF were also carried out under similar reaction conditions (r.t., 2 days, Entries 4-9) for comparison. From these results, it is obvious that the product yields in water are better than that in organic solvents for all three dienes. Table 1. Diels-Alder reaction of cyclohexadienes 1-3 and benzoquinone 4 in various solvents. a

Entry
Although the yield of 7 was lower than that of 5, it was found that the effect of aqueous media was relatively distinct for the reaction of 3 yielding 7. Namely, the yield of 7 in water was nine times higher than that using the organic solvent conditions.
Since dienes 1-3 are insoluble in water, the reaction under neat conditions was examined as a reference (Entries 10-12). The results indicate again that the advantage of water solution was large for precursor 3, which is the least reactive diene among the three. In contrast, no benefit of aqueous media was observed for the reaction of 2, since the adduct 6 was obtained in the same yield under the neat conditions. The relative yields of 5, 6, and 7 in H 2 O/neat conditions were 1.2, 1.0, and 4.0, respectively. The only difference between 2 and 3 is the position of the substituents, with the latter having two substituents on the reactive carbon. This observed increase in yield for 3 but not for 2 can be rationalized as follows (Scheme 2): in the reaction of 3 and 4, the reactants must approach each other as shown in A, which is a 'compact' association and therefore is relatively more favorable in aqueous media than in organic solvents because of the hydrophobic effect. In contrast, the reaction of 2 proceeds through association B, however the hydrophobic effect facilitates the more 'compact' association C. In organic solvents, B is favored over C or A because of steric interactions, which explains the very low yield of 7 in THF or toluene.
Breslow and Rizzol studied the salt effect in the aqueous Diels-Alder reaction of anthracene derivative with ethyl maleiimide, and reported that the reaction is faster after addition of LiCl and slower after addition of guanidium chloride (GnCl) [11,12]. Kumar et al. observed a related phenomenon and explained it in terms of salting-out and salting-in effects [13]. We then examined the salt effect of the present reaction using NaCl and GnCl as the salting-out and salting-in additives [2b], respectively. The effect of NaCl was studied first and the results are shown in Table 2. A distinct retardation was observed for the reaction of 3 (Entries 5,6), while the effect of NaCl was small for both 1 and 2 (Entries 1-4). The yields of the adducts in saturated solution were of a similar value to the case of neat condition, which can be considered as the result of a salting-out effect of water-soluble benzoquinone.
The results in GnCl solution are summarized in Table 3. Here again, the effect of the additive was distinct for the reaction of 3. Namely, in contrast to NaCl, the reaction proceeded smoothly giving the adduct 7 in better yields (Entries 10-12), and after a longer reaction time, 7 was obtained in 76% yield without heating. The yield of 6 was also improved in concentrated GnCl aq. (Entries 6-8), while almost no salt effect was observed for the reaction of 1, except that 5 was obtained in slightly higher yield in concentrated salt solution (Entry 4). These results are in contrast to Breslow's and Kumar's observation, in which the reaction was activated by LiCl and retarded by GnCl, although the substrates and the conditions are different. In order to clarify the role of GnCl, related additives were examined for the reaction of 3 yielding 7. The results are listed in Table 4. When urea, an analogue of guanidium chloride with no charge, was added, the yield of 7 increased (Entries 1-4) but the effect was much smaller than GnCl. Similar results were obtained in acetone (Entries 5-8). These data imply that the simple solvation effect against hydrocarbon 3 is small. A possible explanation of the effect of GnCl is hydrogen-bonding with 1,4-benzoquinone. In mixed solvent system of acetone/water, water is a much important solvent than acetone, since the yield of 7 in acetone without water was only 2%.

Conclusions
It was found that the effect of water in the aqueous Diels-Alder reaction of cyclohexadiene derivatives with 1,4-benzoquinone is more distinct for the compound having two alkyl substituents on the reacting carbon. The effect of an additive was also large for the same compound with regards to both acceleration and retardation. Therefore, the position of alkyl substituents is an important factor in the aqueous Diels-Alder reaction. A favorable compact association was proposed as the explanation.