THE PINEAL GLAND AND CHRONOBIOLOGY
, Head, Section on Neuroendocrinology
Steven L. Coon, PhD, Staff Scientist
Joan L. Weller, BA, Senior Research Assistant
Jong-So Kim, PhD, Visiting Research Fellow
Michael Bailey, PhD, Postdoctoral Fellow
Qiong Shi, PhD, Postdoctoral Fellow
Diego Bustos, PhD, Visiting Fellow
Sam Clokie, PhD,Visiting Fellow
Jiří Pavlíček, PhD, Visiting Fellow
Pascaline Gaildrat, PhD, Guest Researcher
Surajit Ganguly, PhD, Guest Researcher
Estela Muñoz, PhD, Guest Researcher
M.A.A. Namboodiri, PhD, Guest Researcher
Harvey Pollard, MD, PhD, Guest Researcher
Laura Klitten, Special Volunteer1
Martin Rath, Special Volunteer1
Our primary focus is the pineal gland, in particular the regulation of production of the pineal hormone melatonin. Our work has broad implications in vertebrate biology and is of special interest to clinical scientists studying human diseases relating to circadian rhythms, including endocrine pathologies, sleep, mood disorders, and deficiencies in alertness. We have also addressed broader issues, namely, the control of global changes in gene expression that occur in the pineal gland on a 24-hour basis and the establishment of the pineal phenotype during development.
The timezyme—arylalkylamine N-acetyltransferase
We have been at the forefront of investigations into arylalkylamine N-acetyltransferase (AANAT), the second enzyme in the synthesis of melatonin from serotonin (see insert). We recently established that AANAT is critical to the control of the rhythm in melatonin synthesis. In all species examined to date, we observed that the large increase in melatonin synthesis at night causes an increase in melatonin production. Transcriptional mechanisms involved in melatonin synthesis include interactions of cyclic AMP response elements in the AANAT promoter; at the same time, other response elements appear to be involved in the promoter and make expression of AANAT highly tissue-specific. These transcriptional events control both tissue specificity and, in some but not all vertebrates, the rhythmic control of expression of the AANAT gene (Besseau et al., Exp Eye Res 2006;82:620). For example, we determined that rodents experience about a 100-fold increase in the expression of the AANAT gene at night; however, in ungulates and primates, the nocturnal increase is small, demonstrating that transcriptional control mechanisms of melatonin synthesis are not essential in all vertebrates; as discussed below, post-translational mechanisms play an important role.
Figure 5.3
We have also made advances in understanding the post-translational regulation of AANAT. Sequence analysis revealed that all AANATs contain C- and N-terminal sites for phosphorylation by cyclic AMP–dependent protein kinase. We found that the sites are critical in controlling the stability and biological half-life of the molecule. Phosphorylation of the sites controls binding of AANAT to a protective binding partner, 14-3-3 protein, which protects the enzyme from destruction while reversal of the binding can lead to destruction; phosphorylation thus governs the balance between protection and destruction. In collaboration with Fred Dyda and Alison Hickman, we determined the crystal structure of AANAT bound to 14-3-3- proteins and thus established the molecular basis of the interaction. Using whole animals, Surajit Ganguly and Joan Weller demonstrated in a physiological context that the interaction is critical for regulation of the enzyme. Our recent research focused on one of AANAT’s phosphorylation sites, which is located in the C-terminal region. Joan Weller developed highly specific antisera to locate the site, revealing it is phosphorylated at night and that the phosphorylation state of the enzyme decreases immediately after animals are exposed to light. This process is thought to lead to destruction of the enzyme. Surajit Ganguly explored the functional role of phosphorylation of AANAT by establishing a highly sensitive method to measure binding of AANAT to 14-3-3. The results underscored the importance of phosphorylation, emphasized the contribution of the entire AANAT molecule to the binding affinity toward 14-3-3 protein, and established that both the C- and N-terminal PKA sites are important for binding 14-3-3 proteins. An on/off mechanism centered around the C-terminal PKA site switches the enzyme from highly active to highly inactive. In collaboration with Michael Iuvone, we established that the same mechanism operates in the retina to control both AANAT activity and melatonin production (Iuvone et al., Prog Retinal Eye Res 2005;24:433). In the retina, light acts directly on photoreceptor cells to control cyclic AMP and, through this mechanism, controls AANAT activity (Pozdeyev et al., J Neurosci 2006;26:9153). We suspect that light acts through a similar mechanism in photosensitive pinealocytes.
Figure 5.4
In collaborative effort with Philip Cole, Larry Szewczuk, Yousang Hwang, and Weiping Zhen, we used novel synthetic approaches to examine the role of phosphorylation in controlling AANAT. We made AANATs that contain non-hydrolysable phosphate at the N-terminal PKA site and injected them into cells to monitor their biological half-life with antisera. The site prevents destruction of AANAT (Zheng et al., J Biol Chem 2005;280:10462). We also pursued new approaches to the development of AANAT inhibitors in order to identify lead compounds for drug development. The importance of post-translational regulatory mechanisms for human biology is clear from Steven Coon’s studies on the monkey pineal; Coon established that large changes occur in the abundance of AANAT protein and activity but that AANAT mRNA, i.e., gene expression, does not change.
A new line of research, spearheaded by Jiří Pavlíček, focuses on the molecular evolution of AANAT. Pavlíček is examining an important element of the AANAT molecule: a floppy loop—described as Loop 1 in Figure 5.5—that forms a portion of the arylamine-binding pocket. The loop has undergone notable changes during the course of molecular evolution. With Dan Sackett, Pavlíček is studying some of those changes by using a combination of methods to evaluate the importance of specific residues in the floppy loop and to determine their contribution to the greater-than-1,000-fold increase in specific activity of AANAT during chordate evolution. Their work involves analysis of a primitive form of AANAT found in the cephalochorate amphioxus and reflects contributions from Pascaline Gaildrat and Jack Falcón.
Figure 5.5 Organization of AANAT and AANAT Homologues
Humphries A, Wells T, Baler R, Klein DC, Carter DA. Rodent Aanat: intronic E-box sequences control tissue specificity but not rhythmic expression in the pineal gland. Mol Cell Endocrinol 2007;270:43-9.
Hwang Y, Ganguly S, Ho AK, Klein DC, Cole PA. Enzymatic and cellular study of a serotonin N-acetyltransferase phosphopantetheine-based prodrug. Bioorg Med Chem 2007;15:2147-55.
Klein DC. Arylalkylamine N-acetyltransferase: “the timezyme.” J Biol Chem 2007;282:4233-7.
Vuilleumier R, Besseau L, Boeuf G, Piparelli A, Gothilf Y, Gehring WG, Klein DC, Falcón J. Starting the zebrafish pineal circadian clock with a single photic transition. Endocrinology 2006;147:2273-9.
Zilberman-Peled B, Appelbaum L, Vallone D, Foulkes NS, Anava S, Anzulovich A, Coon SL, Klein DC, Falcón J, Ron B, Gothilf Y. Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream. J Neuroendocrinol 2007;19:46-53.
Pineal/retinal evolution, AANAT, and the formation of conjugates of arylalkylamine and retinaldehyde
According to our theory of the evolution of the pineal gland, both the pineal gland and retina evolved from the same primitive photoreceptor cell after that cell acquired AANAT and HIOMT, the enzymes required to produce melatonin. We believe that, originally, the enzymes were important only in detoxification of arylalkylamines; such detoxification, however, can be dangerous in all tissues because of the reactivity of the amine and aldehyde that arises from oxidation of the amine. Detoxification, we posit, led to the production of melatonin and eventually to the development of the rhythm in melatonin as a day/night signal. Our theory proposes that high levels of melatonin were destructive to the primitive photoreceptor; moreover, the required high levels of serotonin, the melatonin precursor, would have been especially toxic to photoreceptor function for the simple reason that the precursor could react with and remove the key photodetection molecule—retinaldehyde. N-bis-retinyl-serotonin (A2S), the theoretical product formed by the reaction, would contain two molecules of retinaldehyde and one of serotonin (Figure 5.6); homologous compounds would be formed from other arylalkylamines, products that belong to a larger family of N-bis-retinyl compounds, including bis-retinal-entholamine. The latter is thought to be toxic to the retina through effects of the retinal side chains. In addition to being toxic, A2S formed in the primitive photoreceptor would reduce photosensitivity by removing retinal. Segregation of the two processes into the pinealocyte and retinal photoreceptor solved the problem of the conflicting processes and made it possible for melatonin production and photodetection to evolve and improve. In collaboration with Ken Kirk, we have synthesized A2S and related compounds and are testing the hypothesis that their formation in the retina is a function of AANAT activity. We are also examining whether the compounds might play a role in human retinal disease, specifically macular degeneration.
Figure 5.6
Coon SL, Klein DC. Evolution of arylalkylamine N-acetyltransferase: emergence and divergence. Mol Cell Endocrinol 2006;252:2-10.
Klein DC. Evolution of the vertebrate pineal gland: the AANAT hypothesis. Chronobiol Int 2006;23:5-20.
Characterization of 14-3-3 proteins in the pineal gland
In collaboration with Alistair Aitken, Qiong Shi used a comparative approach to analyze the isoforms of 14-3-3 proteins, establishing the developmental appearance of each isoform in the pineal gland. The work is based on both mRNA and protein analysis and revealed marked differences in the relative abundance of each isoform. Shi also identified changes in the cellular distribution of some, but not all, isoforms after adrenergic activation. Some forms of 14-3-3 have distinctly greater capacity than others to bind and become activated; using structural and physical biological approaches, in combination with enzymology, we are investigating the molecular basis of differences in binding and activation capacity.
Ganguly S, Weller JL, Ho A, Chemineau P, Malpaux B, Klein DC. Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc Natl Acad Sci USA 2005;102:1222-7.
Global analysis of gene expression
We are pursuing several projects aimed at (1) obtaining a global picture of differences in gene expression that occur on a night/day basis and (2) identifying genes that are highly enriched in the pineal gland. In collaboration with Igor Dawid, we are facilitating the NICHD-sponsored commercialization of an Affymetrix® chip for analysis of gene expression in zebrafish and Xenopus. Together with Dawid, Yoav Gothilf, and Reiko Toyama, we are characterizing pineal gene expression in the zebrafish as a function of time of day and time of development. We are also examining pineal gene expression in other species and, in collaboration with Peter Munson, David Carter, and Ruben Baler, have identified a set of genes highly expressed in the pineal gland. The work provides a basis for analyzing the control of gene expression in second-messenger cascades and has uncovered the expression of genes that previously had not appeared in the pineal literature, such as methionine adenosyltransferase, MAP kinase phosphatase-1, PepT1, phoshodiesterase 4D2, dopamine D4R receptor, and a subunit of the IgE receptor. Identification of new genes in the pineal gland, which are conserved across species, is pointing to new transcriptional pathways controlled by previously unrecognized transcription factors. By analyzing these transcription factors and the promoters of genes that are either upregulated at night or highly expressed in the pineal gland, we will be able to construct a regulatory network that describes the cascade of transcription factors controlling pineal gene expression. The available data indicate that many genes are regulated by a similar mechanism, as illustrated in Figure 5.7.
Figure 5.7
The figure illustrates how light acts through the eye to influence the suprachiasmatic nucleus (SCN), a master oscillator, indicated by the symbol . The neural pathway passes through central neural structures, including the paraventricular nucleus of the hypothalamus (PVN) and the spinal cord, and the peripheral nervous system; the pineal gland is innervated by nerves from the superior cervical ganglia (SCG). The release of the transmitter norepinephrine (NE) from SCG nerves elevates cyclic AMP and intracellular calcium through actions on adrenergic receptors (ARs). Elevation of cyclic AMP acts through cyclic AMP–dependent protein kinase (PKA) to activate cyclic AMP response element–binding protein (CREB), which resides on the promoters of several pineal genes. The result is increased expression of AANAT and other gene products, including PINA, a pineal splice variant form of the ATPase ATP7B; ICER (inducible cyclic AMP early repressor) encoded by the CREM gene; Dio2, Type 2 Diodinase; Fra-2, Fos-related antigen-2; MAT2a; MKP-1, MAP kinase phosphatase-1; pPET1, a pineal product of the gene encoding PepT1 (peptide transporter 1); and RZRβ/RORβ, retinoic acid orphan receptorβ.
MAP kinase signaling in the pineal gland
The norepinephrine-driven increase in mitogen-activated protein kinase (MAPK) activity is part of the mechanism that regulates AANAT activity in the rat pineal gland. In collaboration with Anthony Ho, an expert on MAPK signaling in the pineal gland, we identified in microarray screening a gene encoding a phosphatase that acts on MAPK. We found a very rapid nocturnal increase in the expression of the MAPK phosphatase MAP kinase phosphatase-1 (MKP-1) that was blocked by maintaining animals in constant light or treating them with propranolol (Price et al., FEBS Lett 2004;577). MKP-1 expression was regulated by norepinephrine acting through both alpha- and beta-adrenergic receptors. The results establish that the nocturnal increase in pineal MKP-1 expression in the pineal gland is under the control of a photoneural system. That substrates of MKP-1 can influence AANAT activity suggests the involvement of MKP-1 in the regulation of the nocturnal AANAT signal and melatonin production.
Regulation of S-adenosyl methionine synthesis
Results from our gene-profiling efforts have highlighted the synthesis of S-adenosylmethionine (SAM), the co-factor of the last enzyme in melatonin synthesis. Jong-so Kim determined that expression of the enzyme that produces this co-factor in the pineal gland—methionine adenosyl transferase 2a—increases at night and is accompanied by an increase in enzyme activity and enzyme protein. The molecular basis of the increase involves neural stimulation of the pineal gland by norepinephrine, which results in the elevation of cyclic AMP. The increase in SAM synthesis obviously requires the increased availability of this methyl donor. Regulation of the synthesis of SAM by neural mechanisms has not been previously described; however, the co-factor does play a central role in the synthesis and metabolism of many transmitters (catecholamines, indoles, histamine, and so forth). Accordingly, evidence from the pineal gland that the activity of MAT 2a can be regulated by a neural circuit via a cyclic AMP mechanism suggests that activity of MAT 2a might be regulated by transmitters in other brain regions and that the levels of SAM might be controlled through pharmacological manipulation of MAT 2a expression. The serotonin→melatonin pathway is controlled via the same neural and second-messenger system at two points (Figure 4.8). The system demonstrates strong and rapid control of melatonin production downstream of the enzyme; in fact, the enzyme was previously thought to be the only regulated element of the serotonin-to-melatonin pathway. Thus, it appears that adrenergic cyclic AMP control of melatonin production from serotonin involves at least two targets: AANAT and SAM production.
Figure 5.8
Kim JS, Coon SL, Blackshaw S, Cepko CL, Møller M, Mukda S, Zhao WQ, Charlton CG, Klein DC. Methionine adenosyltransferase: adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression. J Biol Chem 2005;280:677-84.
Regulation of phosphodiesterase 4B2
Using microarray analysis of the pineal gland, Jong-So Kim found that mRNA encoding by the gene PDE4B2 is more than five-fold higher at night than during the day and that expression in the pineal gland is higher than in other tissues, as confirmed by Morten Møller, an anatomist with special expertise in the pineal gland. Kim found that the increase in PDE4B2 mRNA is associated with an increase in protein and activity, which in turn influences the accumulation of cyclic AMP. The same neural pathway that regulates AANAT and MAT controls expression of the PDE4B2 gene; furthermore, cyclic AMP also controls expression of the gene and represents a negative feedback mechanism. The microarray analysis is the first demonstration of a negative feedback mechanism involving cyclic AMP destruction in the pineal gland. The gene also functions as an internal interval timer.
Kim JS, Bailey MJ, Ho AK, Møller M, Gaildrat P, Klein DC. Daily rhythm in pineal phosphodiesterase activity reflects adrenergic/cAMP induction of the PDE4B2 variant. Endocrinology 2007;148:1475-85.
Regulation of acetyl coenzyme A synthesis
We investigated ATP-dependent citrate lyase, the enzyme that regulates formation of acetyl coenzyme A, and found that the pineal expresses particularly high levels of the enzyme as compared with other tissues and that, in some species, the abundance of mRNA encoding the enzyme exhibits daily changes. We found that a physical interaction between ATP-dependent citrate lyase and AANAT brings the source of acetyl coenzyme A directly into contact with AANAT and promotes efficient acetylation of serotonin and melatonin production. It appears that the association might be regulated by phosphorylation and could provide an important element in the general activation of the pineal gland in support of the nocturnal increase in melatonin production.
Induction of the oligopeptide transporter PEPT1
Microarray studies revealed that the expression of the oligopeptide transporter (PEPT1) gene is markedly increased at night (about 100-fold) and that night-time expression of the gene in the pineal gland produces a truncated version of the gene. We also observed that regulation of the gene reflects neural cyclic AMP activation of the pineal gland and noticeably large changes in both the mRNA encoding the PEPT1 protein and the protein itself. This mechanism of regulation of a membrane protein is unusual in that the protein is relatively unstable and disappears rapidly. We also discovered that the expression of the gene product is highly restricted to the pineal gland, and we identified a section of the gene that appears to be responsible for both the pattern of tissue distribution and the night/day pattern of expression. The gene shares features with the AANAT gene with regard to the presence of cyclic AMP response elements and the putative sites—located in an internal promoter—for the binding of CRX/OTX transcription factors.
Gaildrat P, Møller M, Mukda S, Humphries A, Carter DA, Ganapathy V, Klein DC. A novel pineal-specific product of the oligopeptide transporter PepT1 gene: circadian expression mediated by cAMP activation of an intronic promoter. J Biol Chem 2005;280:16851-60.
NeuroD-1, CRX, OTX, Pax4, and Pax6 expression in the pineal gland
Results from microarray analysis revealed that the pineal gland expresses high levels of transcription factors known to play a role in the development of the eye and other tissues: NeuroD1, CRX, OTX, Pax4, and Pax6. In collaboration with Morten Møller’s laboratory, we focused on these five transcription factors to determine whether they are involved in developmental and adult expression of genes in the pineal gland. Radiochemical in situ hybridization and Northern blot analysis established that all are expressed during development and remain at elevated levels in the adult. CRX and OTX2 appear coincidently early in pineal development of the rat and are highly expressed in the adult, apparently to maintain phenotype.
Muñoz EM, Bailey MJ, Rath MF, Shi Q, Morin F, Coon SL, Møller M, Klein DC. NeuroD1: developmental expression and regulated genes in the rodent pineal gland. J Neurochem 2007;102:887-99.
Rath MF, Muñoz E, Ganguly S, Morin F, Shi Q, Klein DC, Møller M. Expression of the Otx2 homeobox gene in the developing mammalian brain: embryonic and adult expression in the pineal gland. J Neurochem 2006;97:556-66.
Studies on the pineal-immune link
Analysis of gene expression in the pineal gland revealed a pronounced daily rhythm in the expression of the gene encoding a subunit of the IgE receptor, reflecting adrenergic/cyclic AMP signaling in pinealocytes. We are attempting to determine the functional role of this rhythm and, to that end, are testing the hypothesis that the pinealocyte serves as a sentinel that can modulate the immune response on a circadian schedule. We also found perivascular phagocytes in the vertebrate pineal gland that serve as antigen-presenting cells. These findings, together with evidence of the expression of the peptide transporter PepT1, provide the initial molecular building blocks of a system mediating the relationship been antigens and the pineal gland.
Ganguly S, Grodzki C, Sugden D, Møller M, Odom S, Gaildrat P, Gery I, Siraganian RP, Rivera J, Klein DC. Neural adrenergic-cyclic AMP regulation of the IgE receptor alpha-subunit expression in the mammalian pinealocyte: a neuroendocrine-immune response link? J Biol Chem 2007 Aug. 29[E-pub ahead of print].
Møller M, Rath MF, Klein DC. The perivascular phagocyte of the mouse pineal gland: an antigen-presenting cell. Chronobiol Int 2006;23:393-401.
Regulation of the dopamine D4R receptor: cAMP + T3 “AND” gate regulation
Preliminary cDNA microarray studies suggested that D4R expression increases at night, which we confirmed with in situ and Northern blot analyses and quantitative PCR. However, we did not find expression above trace levels of other dopamine receptors in the pineal gland. D4R expression in the pineal gland is several-fold higher than in most other tissues, suggesting that the D4R receptor plays an important role in pineal signal transduction, perhaps as an inhibitor of adrenergic stimulation of adenylate cyclase. Compared with other genes, transcriptional regulation of the expression of the D4R gene is unusual. Specifically, in the case of the genes encoding AANAT, MAT2, MKP-1, Type II deiodinase, ICER, and FRA-2, the nocturnal increase in mRNA can be reproduced in organ culture by treatment with either norepinephrine or a cyclic AMP protagonist, but the same does not hold for the D4R receptor. Rather, cotreatment with the thyroid hormone triiodothyronine (T3) and either norepinephrine or cyclic AMP is required to increase D4R mRNA. These findings establish that expression of the D4R receptor is regulated by an “AND” gate operated by cAMP and T3 and provide evidence for a role of the thyroid gland in regulating a specific gene in the pineal gland, consistent with the pineal gland’s increased capacity to convert thyroxin (T4) to T3 through induction of type II deiodinase at night. By suggesting that melatonin synthesis might be a function of thyroid status, our study has broad implications for dopamine signal transduction in other tissues.
Role of islet antigen 2 and islet antigen 2beta in circadian biology
We discovered that the elimination of two proteins, islet antigen 2 and islet antigen 2 beta, leads to the loss of daily rhythms in activity, cardiovascular function, and temperature. The proteins are found in secretory vesicles and are best recognized by their association with Type 1 diabetes; autoantibodies against the proteins are a predictor of the disease. Our studies suggest that the absence of daily rhythms may be attributable to a combination of neurotransmission and downstream effects.
1 Student from University of Copenhagen
2 Fabrice Morin, PhD, former Postdoctoral Fellow
COLLABORATOR
Alastair Aitken, PhD, University of Edinburgh, Edinburgh, UK
Blaine Armbruster, PhD, University of North Carolina, Chapel Hill, NC
Ruben Baler, PhD, Laboratory of Cellular and Molecular Regulation, NIMH, Bethesda, MD
Stefano Bertuzzi, MD, Laboratory of Developmental Neurogenetics, NINDS, Bethesda, MD
Seth Blackshaw, PhD, Harvard Medical School, Boston, MA
David Carter, PhD, University of Wales, Cardiff, UK
Francesco S. Celi, MD, Clinical Endocrinology Branch, NIDDK, Bethesda, MD
Clivel Charlton, PhD, Florida A&M University, Tallahassee, FL
Philippe Chemineau, PhD, Institut National de la Recherche Agronomique, Nouzilly, France
Constance L. Chik, MD, University of Alberta, Edmonton, Canada
Philip Cole, MD, PhD, The Johns Hopkins University, Baltimore, MD
Cara M. Constance, PhD, College of the Holy Cross, Worcester, MA
Igor Dawid, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Fred Dyda, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Jack Falcón, PhD, Observeratoire Océanologique/CNRS, Banyuls, France
Vadivel Ganapathy, PhD, Medical College of Georgia, Augusta, GA
Zoila Garza, MS, Mathematical and Statistical Computing Laboratory, CIT, NIH, Bethesda, MD
Yoav Gothilf, PhD, Tel Aviv University, Tel Aviv, Israel
Sergio A. Hassan, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
Allison Hickman, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Anthony Ho, PhD, University of Alberta, Edmonton, Canada
John Hogenesch, PhD, Genome Institute of Novartis Foundation, San Diego, CA
Ann Humphries, PhD, University of Wales, Cardiff, UK
Yousang Hwang, PhD, The Johns Hopkins University, Baltimore, MD
Ken-Ichi Inui, PhD, Kyoto University Hospital, Kyoto, Japan
P. Michael Iuvone, PhD, Emory University School of Medicine, Atlanta, GA
Howard Jaffe, PhD, Laboratory of Neurochemistry, NINDS, Bethesda, MD
Ken Kirk, PhD, Laboratory of Chemistry, NIDDK, Bethesda, MD
Eugene V. Koonin, PhD, National Center for Biotechnology Information, NLM, Bethesda, MD
Benoit Malpaux, PhD, Institut National de la Recherche Agronomique, Nouzilly, France
Sandford Markey, PhD, Laboratory of Neurotoxicology, NIMH, Bethesda, MD
Morten Møller, PhD, Panum Institutet, Københavns Universitet, Copenhagen, Denmark
Estella Muñoz, PhD, Instituto de Histologia y Embryologia, U.N.Cuyo, Mendoza, Argentina
Peter Munson, PhD, National Center for Biotechnology Information, NLM, Bethesda, MD
Abner Notkins, MD, Oral Infection and Immunity Branch, NIDCR, Bethesda, MD
Tomas Obsil, PhD, Charles University, Prague, Czech Republic
Hugh Piggins, PhD, Manchester University, Manchester, UK
Daniel Sackett, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Jürgen Schnermann, MD, Kidney Disease Branch, NIDDK, Bethesda, MD
Peter W. Schuck, PhD, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD
Peter J. Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
David Sugden, PhD, Kings College, University of London, London, UK
Larry Szewczuk , PhD, The Johns Hopkins University, Baltimore, MD
Reiko Toyama, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
David Weaver, PhD, University of Massachusetts Worcester Campus, Worcester, MA
Al Yergey, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Weiping Zhen, PhD, The Johns Hopkins University, Baltimore, MD
For further information, contact klein@helix.nih.gov.
