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5
Marine Organisms as Models for Biomedical Research

Ought we, for instance, to begin by discussing each separate species…taking each kind in hand independently of the rest, or ought we rather to deal first with the attributes which they have in common in virtue of some common element of their nature, and proceed from this as a basis for the consideration of them separately? (Aristotle, De partibus animalium).

Recognition of the conservation of fundamental processes during evolution requires the comparative study of many different species. As indicated by the quotation from Aristotle introducing this chapter, there is a long tradition behind this comparative approach to biology. The analysis of conserved features has been essential to the study of evolution and the reconstruction of evolutionary relationships. Although the comparative approach emerged from the tradition of natural history, it has also been used extensively in the disciplines of physiology, biochemistry, and developmental biology. Some of the insights gleaned from these studies include the thermoregulatory role of countercurrent exchange systems in the circulatory system, the biochemical evolution of proteins through duplication of structural motifs, and the role of cytoplasmic segregation in the development of embryos. Comparative studies have also helped researchers identify which features of organisms are fundamental to function. The assumption is that a conserved feature is so essential for normal biological processes that any modification would be likely to reduce the viability of the organism.

Even after a critical function has been identified, studies using a diversity of organisms have facilitated the elucidation of mechanisms. Determining the best method for investigating a particular biological phenomena frequently requires choosing the animal model that best lends itself to experimentation. For example,



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Page 83 5 Marine Organisms as Models for Biomedical Research Ought we, for instance, to begin by discussing each separate species…taking each kind in hand independently of the rest, or ought we rather to deal first with the attributes which they have in common in virtue of some common element of their nature, and proceed from this as a basis for the consideration of them separately? (Aristotle, De partibus animalium). Recognition of the conservation of fundamental processes during evolution requires the comparative study of many different species. As indicated by the quotation from Aristotle introducing this chapter, there is a long tradition behind this comparative approach to biology. The analysis of conserved features has been essential to the study of evolution and the reconstruction of evolutionary relationships. Although the comparative approach emerged from the tradition of natural history, it has also been used extensively in the disciplines of physiology, biochemistry, and developmental biology. Some of the insights gleaned from these studies include the thermoregulatory role of countercurrent exchange systems in the circulatory system, the biochemical evolution of proteins through duplication of structural motifs, and the role of cytoplasmic segregation in the development of embryos. Comparative studies have also helped researchers identify which features of organisms are fundamental to function. The assumption is that a conserved feature is so essential for normal biological processes that any modification would be likely to reduce the viability of the organism. Even after a critical function has been identified, studies using a diversity of organisms have facilitated the elucidation of mechanisms. Determining the best method for investigating a particular biological phenomena frequently requires choosing the animal model that best lends itself to experimentation. For example,

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Page 84 the characterization and cloning of the acetylcholine receptor was simplified by the use of the electric organ, a highly modified muscle found in the electric ray Torpedo. Nerve impulses are chemically transmitted to muscles through the acetylcholine receptor, a process disrupted in the human neuromuscular disorder, myasthenia gravis. In the ray's specialized electric organ, this receptor protein is found at such a high density in the cell membrane that researchers were able to determine the structure of the protein and clone the gene without first having to purify the protein. This would not have been possible using human or other mammalian tissue. The validity of applying results obtained from the study of a protein in a highly modified fish muscle to normal biochemical processes in human muscle derives from the insight that many fundamental features at the molecular and cellular level are highly conserved even though the evolution of animals has shown dramatic changes in morphological form (Gerhart and Kirschner, 1997). Studies using marine organisms have had a major influence on biomedical research (Sargent, 1987). This chapter highlights some of the best recognized marine models and elaborates the reasons for their success. However, the first question one might ask is—why marine organisms? At higher taxonomic levels, most biological diversity is found either primarily or exclusively in the ocean. Of 33 modern phyla, only 11 are found in terrestrial habitats while 28 occur in marine habitats. Hence the diversity of life in the sea offers more possibilities for the discovery of organisms for use as models to explore various biological processes. In several of the examples described in this chapter, specific adaptations to the marine environment have been valuable in studying analogous physiological processes in humans. Of particular interest are several marine taxa that share a common origin with mammals. This group, the deuterostomes, includes vertebrates (and other chordates), echinoderms (e.g., sea urchins and sea stars) and tunicates (e.g., sea squirts). Echinoderms appeared early in the fossil record and are the most distant deuterostome relatives of humans. Studies on the differences and similarities of these groups offer insights into the evolution of vertebrates and mammals. Also, many of these organisms have specialized features that have enabled researchers to elucidate complex processes that would be more difficult to study in mammals. A number of examples are listed in Table 5-1, of which some are described in more detail in this chapter. Sea Stars, Sea Urchins, Tunicates, and Sharks: Their Role in Understanding How the Body Fights Infection and Disease In 1882, Elie Metchnikoff conducted an experiment with sea star larvae (Beck and Habicht, 1996). He punctured a larva with the thorn of a rose and the next day observed tiny motile cells surrounding the thorn. He postulated that the motile cells were a defense mechanism against foreign invaders. The process

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Page 85 TABLE 5-1 Examples of Marine Species Used in Biomedical Research General Human Health Issue Taxon Species (Common Name) Human Medical Concern Immunology Mollusca Conus spp. (cone snails) Blood disorders, clotting, hemophilia   Echinodermata Arbacia punctulata (sea urchin) Cell-mediated immune system responses   Tunicata Botryllus schlosseri (Golden Star Tunicate, sea squirt) Immune systems and disorders (self/non-self recognition), AIDS/HIV transmission   Vertebrata, Fish Squalus acanthias (spiny dogfish shark) Immune system function, evolution of antibodies, and disease resistance Neurobiology Mollusca Loligo pealei (squid) Aplysia (marine snail) Neurological studies, behavior Nerve impulse transmission   Arthropoda Limulus polyphemus (horseshoe crab) Vision Neural basis of behavior   Vertebrata, Fish Opsanus tau (toadfish) Balance and equilibrium, nausea     Squalus acanthias (spiny dogfish shark) Brain function Vision, glaucoma, cataracts     Pomacentrus partitus (bicolor damselfish) Neurofibromatosis     Electrophorus electricus (electric eel) Synaptic transmission (NA+ K+ ATPase) Cell Biology/ Cancer Mollusca Spisula solidissma (surf clam) Cell division/cancer     Loligo pealei (squid) Cell physiology, intracellular transport, and cellular pH calcium regulation   Echinodermata Arbacia punctulata (sea urchin) Cell division/cancer       Fertilization and development   Arthropoda Cancer irroratus (red crab) Organic ion transport   Vertebrata, Fish Pseudopleuronectes americanus (winter flounder) Organic ion transport Physiology Arthropoda Eriocheir sinensis (Chinese mitten crab) Cellular osmoregulation     Cancer irroratus (red crab) Detoxification mechanisms     Carcinus maenas (green shore crab) Kidney function   Vertebrata, Fish Opsanus tau (toadfish) Insulin secretion and diabetes       Muscle pathologies     Squalus acanthias (spiny dogfish shark) Cystic fibrosis Kidney and heart research     Pseudopleuronectes americanus (winter flounder) Detoxification mechanisms     Anguilla rostrata (American eel) Kidney function

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Page 86 Metchnikoff observed was phagocytosis. Although phagocytosis had already been observed with human cells, his observations led him to suggest that the process might be a more fundamental defensive mechanism that is widespread in the animal kingdom. Further research showed that echinoderms (e.g., sea urchins and sea stars) possess the features of a basic immune system, one that involves the non-specific action of phagocytic cells (Smith and Davidson, 1994). This is believed to be the oldest form of immunity and, as suspected by Metchnikoff, it appears to be shared by all animals. Metchnikoff's work on sea stars laid the foundation for the disciplines of cellular and comparative immunology and all subsequent studies on the role of cells and phagocytosis in fighting infection and disease in humans.1 Further insight into how the human immune system functions has been obtained from studies of other marine deuterostomes. Tunicates, or sea squirts as they are commonly called, have provided a model for studying another aspect of immunity, the ability to distinguish ''self" from "non-self." When two sea squirts come into contact they will fuse into one organism if related or grow apart if they are unrelated. Observations of this phenomenon in the field led to the laboratory use of tunicates as a model system for tissue transplantation studies (Raftos, 1994). Tissue recognition or rejection is mediated by the immune system. In sea squirts and humans, similar strategies have been described for determining tissue compatibility involving specialized cells and specific self-recognition molecules. The recognition of "self" is also important to the reproduction of sea squirts. These animals are hermaphrodites, meaning that the same individual can produce both sperm and eggs. However, there is no fertilization of eggs by sperm produced from the same animal. Studies of the tunicate Botryllus schlosseri also showed that the sperm from one individual does not bind to blood cells from the same individual, although they do bind to blood cells from a different individual. This observation prompted some AIDS researchers to conduct a similar experiment with human sperm and blood cells (Scofield, 1997). They found that human sperm, like tunicate sperm, exclusively bind to blood cells from other individuals and discriminate through detection of a self-recognition molecule on the surface of the blood cells (Scofield et al., 1992). This discovery may play a role in understanding how the AIDS virus is transmitted and help biomedical researchers devise protocols for reducing disease transmission. The phagocytic cells of marine invertebrates constitute the most fundamental type of animal immune system. The response is rapid and is referred to as natural or innate immunity. Vertebrates possess an additional form of immunity referred to as acquired or adaptive immunity. This form of immunity relies on a combinatorial genetic mechanism that generates millions of specific recognition molecules in specialized defense cells, the B and T lymphocytes. Insight into the evolution 1 The importance of Elie Metchnikoff's work was recognized in 1908 when he was awarded the Nobel Prize for Physiology or Medicine.

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Page 87 of this important feature of the human immune system has come from the study of sharks. Sharks and other cartilaginous fishes are the most primitive group of vertebrates with this adaptive, combinatorial immune system. Sharks first appeared in the fossil record between four and five hundred million years ago and their long history suggests that this "new" immune system gave sharks an evolutionary advantage which allowed them to survive while other taxa became extinct (Litman, 1996). The immune system of sharks shares some similarities to the human fetal immune system because the predominant circulating class of anti-bodies in the shark resembles the earliest produced immunoglobulin M (IgM) macroglobulins of fetal humans. Sharks also possess innate anti-microbial anti-bodies, T-cell receptors, and major histocompatability antigens (MHCs). Thus, sharks present a comparative model for studying both innate and acquired immunity and autoimmunity, which is the underlying cause of several human diseases such as lupus and rheumatoid arthritis. In addition, sharks possess the steroid squalamine which has an immunomodulatory function and antimicrobial activity with pharmaceutical potential (Moore et al., 1993). Although many aspects of the immune system can be studied in mammals, studies that have taken a comparative approach have provided valuable insight into the basic mechanism of immune responses. The comparative approach may hold the key to developing new therapies for autoimmune and immunodeficiency diseases (Marchalonis and Schluter, 1994). Sea Urchin and Clam Eggs: Their Role inUnderstanding Cell Biology and Biochemistry Sea urchins have served as experimental models for more than 100 years. Many species produce tremendous quantities (millions to billions; Plate XV) of large, clear eggs that lack external coatings. An early example of their utility was Otto Warburg's demonstration in 1908 of the increase in oxygen consumption that occurs following fertilization, despite the relative insensitivity of his methods. The cell cycle is the orderly sequence of events in which a cell first reproduces its genetic material and then divides. In the life of an organism, cell division begins following the fertilization of the egg, defines the early growth and differentiation of the embryo, and continues throughout adulthood, especially in tissues like blood and intestinal mucosa. The cell cycle is closely regulated by a group of proteins, the cyclins (Pines, 1996), which were originally identified in sea urchins (Evans et al., 1983). The key features of sea urchin eggs that made this discovery possible are their abundance and the synchronous division of cells after fertilization. Researchers radiolabeled newly fertilized eggs to tag proteins synthesized during the first few cell divisions. They found that while most proteins accumulate through succeeding cell cycles, one protein, cyclin, is remarkable in that it is synthesized and destroyed once per cell cycle, appearing and disappearing periodically as the cell divides.

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Page 88 Cyclin A was first cloned from the surf clam and its connection to the cell cycle was confirmed by its ability to initiate meiosis in frog eggs and mitosis in somatic cells. Subsequent studies have shown that the synthesis and destruction of cyclins are the key events in the regulation of cell division in all eukaryotic cells, from yeast to human. Clued by the sequences of the cyclins discovered in marine invertebrates, scientists have identified many different cyclins in mammalian cells. Currently, there is considerable excitement among cancer researchers following the finding that in many human cancers there are mutations that change the function of particular cyclins or the proteins that either regulate or are regulated by cyclin (Hall and Peters, 1996). Thus, the discovery of cyclins in sea urchin eggs revolutionized the study of the mammalian cell cycle and paved the way for new research into the diagnosis and treatment of cancer. Transient and localized changes in intracellular calcium concentration are ubiquitous signals for many essential cellular responses (Lee, 1997). Sea urchin eggs are the preferred model for investigating calcium signaling because: (1) the eggs are large, transparent, and amenable to microinjection, allowing localized changes in calcium concentration to be readily visualized using dyes and (2) the abundant cytoplasm can be harvested and fractionated to identify the interacting cellular components of the signaling pathway. More than 50 years ago a spike in intracellular calcium concentration was identified in Arbacia eggs following fertilization. This calcium signal triggers formation of a clear proteinaceous fertilization envelope that surrounds the entire egg, and by preventing sperm from reaching the egg membrane, acts as a mechanical block to polyspermy. The calcium signal also contributes to the signal to activate protein and DNA (deoxyribonucleic acid) synthesis at the onset of development. Curiously, the calcium signal occurs as a wave that begins at the site of sperm-egg fusion and sweeps across the entire egg in approximately 30 seconds (Plate XVI). Such waves, which can be repetitive, occur in many different kinds of cells in response to a wide variety of stimuli. The nature of the waves has been elucidated by the discovery that they depend on chemical intracellular messengers, some of which were only recently identified in an exciting series of investigations using sea urchin eggs (Lee, 1997). The calcium that constitutes these waves is released locally from intracellular stores identified as the endoplasmic reticulum. The initial signal, calcium-induced calcium release, propagates the waves by successively stimulating calcium receptors in adjacent intracellular membranes, resulting in further calcium release. Based on new results using sea urchin eggs, there are at least two separate calcium stores, each with a specific receptor selectively sensitized by one of two novel endogenous chemicals, cyclic adenosine 5'-diphosphate-ribose (cADP-ribose) and nicotinic acid adenine dinucleotide phosphate (NAADP). The cADP-ribose and NAADP were identified by analysis of sea urchin egg cytoplasm.

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Page 89 Additional proteins that modulate cADP-ribose and NAADP activity were also identified in the egg cytoplasm. The importance of these findings is highlighted by the discovery that numerous types of mammalian cells are responsive to cADP-ribose, including neurons and muscle cells in heart, intestine, and skeletal muscle. The cADP-ribose acts on ryanodine receptors that couple increases in intracellular calcium concentration to muscle contraction. Cyclic ADP-ribose also accounts for the calcium-mobilizing action of the nitric oxide signaling pathway. Thus, the cADP-ribose system that was only recently discovered as a result of continuing investigations of sea urchin eggs appears to be of fundamental importance for mammalian neuromuscular coordination and, with further research, should contribute to advances in the diagnosis and treatment of neuromuscular disorders. Marine Organisms: Their Role in Physiological Studies Pertaining to Fluid and Ion Transport, Renal Function, and Volume Regulation Marine organisms have proven the value of the August Krogh principle, which essentially states that for every problem in physiology, there is one animal ideally suited to solve that problem. In particular, marine invertebrates and fishes have been important as models for osmoregulatory phenomena such as fluid and ion transport, renal function, and volume regulation. It is important to recognize why marine organisms have developed extensive osmoregulatory capabilities in order to appreciate how the principles learned from studying osmoregulation in marine animals have led to an increased understanding of osmoregulatory phenomena in humans. Simply, osmoregulation is necessary for the survival of animals in salt-variable environments ranging from estuaries and mangrove swamps to the Dead Sea. Marine invertebrates and fishes are exposed to salinities as high as 2.5 times that of normal seawater or 2500 mOsm ("milliosmoles/liter," a term used to quantify the total concentration of osmotically active solutes in a solution). A parallel situation occurs in humans; the mammalian extracellular fluid is regulated at about 300 mOsm but some mammalian kidney cells are exposed to concentrations as high as 3000 mOsm. Most marine organisms have permeable tissues that are in direct contact with their environment. Two examples will illustrate the physiological problems that this generates. The first is an osmoconformer, or an organism whose body fluids are the same salt concentration as the seawater that surrounds them. As the salt concentration of the water rises, so does the salt concentration of its body fluids. This causes cellular shrinkage which, if uncorrected, would ultimately result in cell death. Instead, a process called volume regulation takes place. The second example is an osmoregulator, or an organism whose body fluids are maintained at a fixed concentration regardless

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Page 90 of the salt concentration of the seawater. A fish or a crab can maintain a blood salt concentration of 300 mOsm. However, gills and portions of the gut are bathed in seawater that is 1000 mOsm. These surfaces are permeable, allowing the entry of salts and the escape of water. This presents two problems: (1) regulating cell volume and (2) restoring body fluid concentration through osmoregulatory mechanisms. In a similar manner, components of both of these processes function in many human organs and, in particular, in the mammalian kidney. Osmoregulation at the intra- and extracellular level is made up of the combined mechanisms of fluid and ion transport and solute or organic osmolyte regulation. The principles learned from studying osmoregulation in marine animals has increased the understanding of how the human kidney maintains the blood at 300 mOsm and also how some kidney cells tolerate the osmotic stress generated by the kidney's role in concentrating urine. With the exception of the halobacteria, the cells of all organisms have the same adaptive response to a high salt environment. They accumulate intracellular organic osmolytes. It was in the Chinese mitten crab, Eriocheir sinensis, that in 1955, (Duchâteau and Florkin, 1955) intracellular accumulation of amino acids with increased salinity was first discovered. Various organisms accumulate solutes intracellularly, but these compounds fall into only a few chemical categories (Yancey et al., 1982). They are polyols, methylamines, and amino acids that help compensate for the high salt environment. This brings up the obvious question of whether there is some highly conserved mechanism that organisms have in common, or whether the similarity in adaptive response is the result of convergent evolution. The hypothesis that there is a conserved mechanism led to the discovery of an osmotic response element (ORE) (Ferraris et al., 1994, 1996) in the flanking region of the aldose reductase (AR) gene, which is responsible for the adaptive accumulation of a type of polyol during high salt stress. The ORE has also been found in other genes and in other species (Ruepp et al., 1996; Takenaka et al., 1994). Hence studies that started with the Chinese mitten crab have led to the discovery of the osmoregulatory function of aldose reductase and subsequently to the finding that inappropriate expression of the same gene in the eye and nerve causes serious damage in diabetic patients. Knowledge of the aldose reductase gene is currently being used to determine the mechanism of inappropriate genetic expression in diabetes. There are several other examples where studies of properties of marine organisms have helped elucidate the mechanisms underlying a variety of biomedical problems caused by defects in fluid or ion transport. Several of these studies are described below. The kidney proximal tubules of Pseudopleuronectes americanus, the winter flounder, and the urinary bladder of Cancer irroratus, the common red crab, have been used as models to examine mechanisms of organic anion transport (David S. Miller, National Institute of Environmental Health Sciences). Organic ion transport is the cellular mechanism by which kidney cells transfer potentially toxic

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Page 91 compounds from the blood into the urine. These compounds include drugs, normal and drug metabolites, as well as environmental pollutants and their metabolites. The gulf toadfish, Opsanus beta, has been used for studies on the regulation of nitrogen metabolism and urea excretion. Most fish excrete nitrogen waste as ammonia, a toxic compound that is rapidly diluted in water. However, toadfish have the capability of switching to secreting urea, a much less toxic metabolite. Urea excretion increases as a response of the fish to confinement and crowding (Walsh et al., 1994) and appears to be regulated by mechanisms similar to thos regulating urea transport in the mammalian kidney (Wood et al., 1998). The eye of Squalus acanthias, the spiny dogfish shark, is used to understand mechanisms of vision and fluid formation, with relevance for human diseases that affect intraocular pressure (like glaucoma) and lens opacities. The rectal gland of Squalus acanthias, the major salt secreting organ in the shark, has proven to be an ideal model system; atrial natriuretic peptides isolated from the dogfish heart, have been shown to control sodium chloride excretion from the rectal gland in combination with vasopressin (Silva et al., 1996). Atrial natriuretic peptide and vasopressin also regulate salt and water excretion by the human kidney. Further, because of the unusually high density of receptors and channels in the shark rectal gland, it has provided an excellent system for research on the regulation of chloride secretion in higher vertebrates, including humans (Forrest, 1996). The gills of marine organisms form a key interface between the blood and the environment. The gills of Carcinus maenas, the green shore crab have provided a useful model for understanding the regulation of analogous sodium transporters found in the mammalian kidney (Towle et al., 1997). The gills of Anguilla rostrata, the American eel, have provided a model system to study how restructuring of the plasma membrane allows kidney cell membranes to adapt when salinity changes (Crockett et al., 1996). The Toadfish: Its Role in Unraveling the Neural Control of Balance and Equilibrium The maintenance of balance and equilibrium in vertebrates is controlled by the vestibular apparatus (the anatomical structures concerned with the vestibular nerve, a somatic sensory branch of the auditory nerve) and its proper functioning is critical for most organisms including humans (Kornhuber, 1974). The vestibular system of toadfish has been used for decades as a model for studying balance and equilibrium. The toadfish was initially chosen for study because it has a broad flat head that makes it relatively easy to study the brain and nerves associated with the vestibular system, and the fish are easy to obtain and adapt readily to the laboratory. This vestibular system is composed of fluid-filled canals lined with small hair cells that sense movement of small crystals, the otoliths. The

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Page 92 hairs were first studied in the early 1900s by Cornelia Clapp at the Marine Biological Laboratory in Woods Hole, MA. When the head moves, the otoliths move, and the hair cells send this information to the brain. Vestibular systems developed early in the evolutionary history of vertebrates and did not change greatly as new species evolved. Thus the vestibular system of the toadfish is homologous to the vestibular system in humans and can be used to better understand the basis for human balance disorders. Horseshoe Crabs: Their Role in Understanding Retinal Function and How Eyes See Knowledge of human vision has its roots in early 20th century studies of the compound eye of the horseshoe crab and the phenomenon of lateral inhibition (Sargent, 1987). The horseshoe crab eye has approximately 1000 photoreceptors whereas the human retina has more than 100 million photoreceptors. Photoreceptors perceive and process visual information about the external environment and then transfer this information to the brain. Hence, studies of photoreceptors and retinas are relevant to understanding the neural basis of behavior. Horseshoe crabs and other marine organisms are particularly amenable to research on retinas because the tissue is readily accessible and can be removed from the animal and studied in the laboratory for long periods of time. The neural network in the horseshoe crab lateral eye is large, there is a cell-based model of this network, and the animal's behavior in the field is well known (Passaglia et al., 1997). Further work on the horseshoe crab may hold the key for deciphering the neurological basis for vision. Aplysia: Its Role in Discovering the Molecular Basis of Learning and Memory The marine snail Aplysia has been a valuable model in studies of neurobiology and behavior. It has a simple brain, made up of only 20,000 nerve cells, in contrast to the mammalian brain that comprises around a trillion cells. Moreover, the Aplysia nerve cells are large and have characteristic locations, which allow them to be identified in each individual. This allows the comparison of a nerve cell in the experimental animals—or those that have learned a response—with the functionally identical nerve cell in the untrained control animals. Initial studies delineated a simple behavior, the gill-withdrawal reflex, and analyzed its neural circuit (Bailey, C.H. et al., 1996; Kandel et al., 1986). It turned out that even this very simple reflex can be modified by three different elementary forms of learning—habituation, sensitization, and classical conditioning. These forms have similar counterparts in humans. Furthermore, each of these forms of learning gives rise to both short-term memory (lasting minutes) and long-term memory (lasting days to weeks) depending upon the number of training trials. This work

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Page 93 provided the first evidence that learning and memory storage involve changes in strength of synaptic connections made between nerve cells of the brain. It also showed that a single synaptic connection can participate in different learning processes and can, in fact, be modified in different ways by these different learning processes (Bailey, C.H. et al., 1996; Kandel et al., 1986). Thus, a single synapse can serve as a site for more than one type of memory storage and these synaptic storage sites have remarkable flexibility. Subsequent studies elucidated the biochemical mechanisms of memory storage. The investigators began with a short-term form of learning, sensitization, and studied its effects on one readily analyzable component of the gill-withdrawal reflex, namely the synaptic connections between the sensory and motor neurons. They discovered that the transient strengthening of the synaptic connections produced by learning involves an increase in transmitter release. This increase is maintained for the duration of the learned response (memory). The biochemical process underlying memory depends on the recruitment of a major intracellular signaling pathway, the cAMP and cAMP-dependent protein kinase pathway. These studies provided the first biochemical insights into the molecular events for short-term memory storage and illustrated that maintenance of memory is the result of the persistence of the signal transmitted by cAMP (Silva et al., 1998). The conversion of transient, short-term memory to a self-sustained, long-term memory was also studied in Aplysia. The switch from short- to long-term memory begins with the movement to the cell nucleus of the cAMP-dependent protein kinase. Here, this signaling kinase coordinates the activation of a number of genes by turning on CREB-1, an activator of gene expression, and by turning off CREB-2, a repressor of gene expression. With the activation of CREB-1, new genes are transcribed that initiate the growth of new synaptic connections (Silva et al., 1998). Current studies on mammals confirm that the same principles pertain to their learning and memory. Thus, the ground breaking studies on Aplysia have led directly to a molecular understanding of learning and memory and provide an avenue to more effective treatments of cognitive disorders (Bailey, C.H. et al., 1996; Kandel et al., 1986). The Squid Giant Axon: Its Role in Establishing How Nerve Impulses are Conducted The discovery of the squid giant axon opened up new avenues of neurobiological research (Baker, 1984; Hodgkin, 1958). The axon, which is 0.5 mm in diameter or 1000-fold larger than vertebrate axons, was identified in 1909 by L.W. Williams, who noted that, "the very size of the nerve processes has prevented their discovery, since it is well-nigh impossible to believe that such a large structure can be a nerve fiber." In 1936, J. Z. Young dispelled the critics through

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Page 94 his demonstration of an action potential following stimulation of the giant axon with a crystal of sodium citrate. Before 1939, nerve action potentials were measured indirectly by applying electrodes to the outside of nerves, which gave very limited information. In 1939 Alan Hodgkin and Andrew Huxley began experimenting with squid giant axons at Plymouth.2 First, Huxley inserted a needle directly into a fiber, intending that they should measure the viscosity of the cytoplasm. When that did not work, Hodgkin and Huxley inserted a glass electrode into the fiber and directly measured the electrical change when a nerve impulse passed. The resting potential was 50 mV negative relative to the surrounding seawater, as expected, but when the fiber was stimulated, the internal potential reached 50 mV positive. Hodgkin and Huxley continued their work on the squid axon, showing that the action potential was propagated by sequential changes in sodium and potassium ion conductance in the membrane throughout the length of the axon. Later investigations showed that nerve conduction is essentially the same in vertebrate neurons as in squid axons. The proteins that form the channels responsible for the sodium and potassium conductance have been cloned and examined in molecular detail, leading to an understanding on an atomic level of the mechanism of conduction. Throughout these studies the squid giant axon remained the preferred system, revealing, for example, the "gating currents" responsible for the changes in conductance that occur during the action potential (Keynes, 1983). Because of their large size, it is relatively easy to insert electrodes of all sorts directly into the neurons without significant damage and it is possible to collect cytoplasm for biochemical analysis. This has led to many other breakthroughs, including pioneering studies in the regulation of intracellular calcium levels and pH. These revolutionary studies on nerve conduction made possible by the use of this marine model form the foundation for current research in neurophysiology, and the concepts that have emerged are the basis for diagnosis and treatment of disorders of conduction in nerves and other excitable tissues such as heart and skeletal muscle. Use of Fish as Models for Human Diseases Fish offer an alternative to rodents for exploring mechanisms of environmental carcinogenesis. In particular, George Bailey's laboratory at Oregon State University has promoted the use of the rainbow trout as a model for research on compounds that cause cancer. This fish offers the advantages of low rearing costs, an ultrasensitive bioassay using abundant embryos whose small size permits observation of tumor development at minute doses of carcinogen, sensitivity 2 Alan Lloyd Hodgkin and Andrew Fielding Huxley won the Nobel Prize for Physiology or Medicine in 1963 for their work with the giant squid axon.

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Page 95 to many classes of carcinogens, well-described tumor pathology, and responsiveness to tumor promoters and inhibitors (Bailey, G.S. et al., 1996). The bicolor damselfish, Pomacentrus partitus, is the first animal model for one type of human cancer that attacks the nervous system, neurofibromatosis type 1. This cancer is found in natural populations of damselfish and has been demonstrated to be transmissible by injection of extracts of cultured tumor cells. This observation led to the isolation of a retrovirus, possibly the causative agent for damselfish neurofibromatosis (Schmale et al., 1996). Conclusions Although some of the most familiar marine animal models were developed early in this century, the promise of using these organisms in research is rediscovered roughly every decade (NIEHS News, EHP 102:272). The discovery of marine organisms as useful models for biomedical research has frequently been serendipitous, but many successful marine models have emerged from a thorough understanding of the natural history and basic biology of marine organisms. Strategies for supporting biomedical research through the use of marine models include: • Encourage education and research in natural history, taxonomy, physiology, and biochemistry of marine organisms as the foundation for the development of valuable new models for biomedical research. As science, and biology in particular, grows more and more compartmentalized into sub-disciplines, it becomes increasing important to foster interdisciplinary approaches to biomedical problems through educational and research opportunities. • Promote the development of nonmammalian models for biomedical research and laboratory culture of targeted marine species. The Comparative Medicine program at the National Center for Research Resources at the NIH currently supports research to explore and develop alternative animal models for biomedical research and funds centers that supply laboratory reared Aplysia and various cephalopods (squid, cuttlefish, and octopus). This type of program is important because it provides researchers with a dependable source of experimental animals that can be bred to both reduce individual variability and to develop new genetic strains. Also, collection of marine animals from natural populations in some cases risks the depletion of the species in the wild. Development of culture techniques and facilities for marine organisms will give more alternatives to the use of mammals in research and will promote our understanding of complex biomedical problems.

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