Hypoxia tolerance in animals: biology and application.

Many invertebrates and ectothermic vertebrates successfully cope with a fluctuating supply of ambient oxygen-and consequently, a highly variable tissue oxygenation-through increasing their antioxidant barriers. During chronic deprivation of oxygen, however, the hypometabolic defense mode of the fruit fly Drosophila, the hypoxia-induced behavioral hypothermia of the crayfish Pacifastacus leniusculus, and the production of ethanol during anoxia by the crucian carp Carassius carassius all indicate that these animals are also capable of utilizing a suite of genetic and physiological defenses to survive otherwise lethal reductions in tissue oxygenation. Normally, much of an organism's gene response to hypoxia is orchestrated via the hypoxia-inducible transcription factor HIF. Recent developments expand our view of HIF function even further by highlighting regulatory roles for HIF in the hypometabolism of insects, in the molting and the normoxic immune response of crustaceans, and in the control-via the downstream effector gene erythropoietin-of the hypoxic ventilatory response and pulmonary hypertension in mammals. These and related topics were collectively presented by the authors in a symposium of the 2008 ICA-CBP conference at Mara National Reserve, Kenya, Africa. This synthesis article communicates the essence of the symposium presentations to the wider community.

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Evolution of novel functions: cryptocyanin helps build new exoskeleton in Cancer magister.

Hemocyanin, the blue blood protein of many arthropods and molluscs, reversibly binds oxygen at its highly conserved copper-oxygen-binding sites and supplies tissues with oxygen. Cryptocyanin, closely related structurally and phylogenetically to arthropod hemocyanin, lacks several of the six critical copper-binding histidines, however, and has lost the ability to bind oxygen. Despite this loss of function, cryptocyanin continues to be synthesized, an indication that it has been exploited to carry out new functions. Here, we show that cryptocyanin is present in extremely high concentrations in the hemolymph of the crab during the premolt portion of the molt cycle. Both proteins are specifically expressed in the same type of cell in the hepatopancreas and secreted into the hemolymph, but cryptocyanin plays a major role in forming the new exoskeleton, while hemocyanin functions in oxygen transport. A cessation in cryptocyanin, but not hemocyanin, synthesis after eyestalk ablation supports our hypothesis that cryptocyanin is closely regulated by molting hormones. The contrasts between the two gene products illustrate how a gene duplication of a copper-oxygen protein and its subsequent mutation may work in concert with the evolution of new regulatory mechanisms, leading to the assumption of new functions.

SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister.

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.

Ontogeny of decapod crustacean hemocyanin: effects of temperature and nutrition.

Hemocyanin is present throughout the decapod crustacean's life, usually as one-hexamer and two-hexamer oligomers. Hemocyanins of some decapod crustaceans undergo changes in subunit composition and oxygen affinity during development. Maternal hemocyanin is taken up from the hemolymph via endocytosis by the oocyte. Embryo hemocyanin differs in subunit composition from hemocyanin of oocyte and adult crab and may represent the onset of hemocyanin synthesis. Complex changes in expression of hemocyanin subunits occur through megalopa and early juvenile stages of the crab Cancer magister, culminating in the pattern of adult hemocyanin. The influences of food availability and temperature on development, growth and hemocyanin ontogeny in early juvenile C. magister have been studied. Crabs were raised in warm or cold sea water and fed high or low levels of food for 6 months. While intermolt period was shorter in crabs fed high food levels, especially those raised in warm water, crabs reared in cold water with high food levels attained the largest sizes. Thus increased food availability affects growth more than increased temperature. Adult hemocyanin appeared at about the same number of weeks after the start of the experiment for crabs in the warm water/high food, warm water/low food and cold water/high food groups, even though warm water/low food crabs had molted fewer times. Crabs in the cold water/low food group expressed adult hemocyanin much later than the other groups. Molt stage and maturation from juvenile to adult are not absolutely coupled, and food availability has a greater influence than temperature on hemocyanin ontogeny.

Cops and robbers: putative evolution of copper oxygen-binding proteins.

Two closely related copper proteins, phenoloxidase and haemocyanin, are known to be involved in different physiological functions such as the primary immune response and oxygen transport. Although the proteins differ structurally, they have the same active site by which dioxygen is bound. Recent results reveal that haemocyanin also exhibits phenoloxidase activity. A scenario is proposed for the evolutionary relationships among copper oxygen-binding proteins (COPs).

Cryptocyanin, a crustacean molting protein: evolutionary link with arthropod hemocyanins and insect hexamerins.

Cryptocyanin, a copper-free hexameric protein in crab (Cancer magister) hemolymph, has been characterized and the amino acid sequence has been deduced from its cDNA. It is markedly similar in sequence, size, and structure to hemocyanin, the copper-containing oxygen-transport protein found in many arthropods. Cryptocyanin does not bind oxygen, however, and lacks three of the six highly conserved copper-binding histidine residues of hemocyanin. Cryptocyanin has no phenoloxidase activity, although a phenoloxidase is present in the hemolymph. The concentration of cryptocyanin in the hemolymph is closely coordinated with the molt cycle and reaches levels higher than hemocyanin during premolt. Cryptocyanin resembles insect hexamerins in the lack of copper, molt cycle patterns of biosynthesis, and potential contributions to the new exoskeleton. Phylogenetic analysis of sequence similarities between cryptocyanin and other members of the hemocyanin gene family shows that cryptocyanin is closely associated with crustacean hemocyanins and suggests that cryptocyanin arose as a result of a hemocyanin gene duplication. The presence of both hemocyanin and cryptocyanin in one animal provides an example of how insect hexamerins might have evolved from hemocyanin. Our results suggest that multiple members of the hemocyanin gene family-hemocyanin, cryptocyanin, phenoloxidase, and hexamerins-may participate in two vital functions of molting animals, oxygen binding and molting. Cryptocyanin may provide important molecular data to further investigate evolutionary relationships among all molting animals.

Functional adaptations of oxygen-transport proteins.

Oxygen-transport proteins are multisubunit, circulating molecules that provide an efficient supply of oxygen to metabolically active metazoans. Hemoglobins, hemerythrins and hemocyanins have evolved in both structural and functional diversity and exhibit functional repertoires beyond that of simple, monomeric tissue myoglobins. Their phylogenetic distribution is intriguing, especially with respect to those organisms that express more than one type of oxygen-transport protein. An animal can modify the delivery of oxygen to its tissues by varying the rate of synthesis of these proteins or by selective expression of individual subunits and/or molecules. Changes in levels of allosteric modifiers that affect the protein's oxygenation properties will also modify oxygen delivery; some organisms have more ability than others to control concentrations of modulators. Hemoglobins have assumed functions in addition to oxygen transport, while hemocyanins have diversified through multiple gene duplications and functional specializations. Understanding the mechanisms of regulation of expression, synthesis and modulator levels is a key focus of current investigations.

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family.

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer-type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.

Developmental changes in hemocyanin expression in the Dungeness crab, Cancer magister.

The copper-based respiratory protein hemocyanin undergoes a developmental shift in subunit composition and function analogous to that seen in many hemoglobins. We studied hemocyanin gene expression in the Dungeness crab (Cancer magister) by Northern blot analysis. Animals were raised under controlled conditions, and total RNA was isolated from 13 developmental stages as well as from six tissue types in the adult animal. RNA was run on formaldehyde-agarose gels, blotted onto nylon membranes, and probed with 32P-labeled cDNA probes specific for C. magister adult hemocyanin. Results indicate that adult hemocyanin biosynthesis occurs in hepatopancreas tissue only. Analysis of developmental stages shows that expression of adult-type hemocyanin, as indicated by the appearance of hemocyanin subunit 6 mRNA, begins during the sixth juvenile instar.

Hemoglobin Structure and Function in the Rat-Tailed Sea Cucumber, Paracaudina chilensis.

The rat-tailed sea cucumber, Paracaudina chilensis, has abundant hemoglobin-filled hemocytes in its perivisceral coelom, water vascular system, and hemal system. The perivisceral oxyhemoglobin consisted of 34 kDa dimers and molecules with an apparent molecular weight of ca. 50 kDa. The perivisceral hemoglobin had a high oxygen affinity with a P50 of 1.5 mm Hg at 15°C. It exhibited cooperative oxygen binding with a Hill coefficient of 1.26 to 1.86. Oxygen affinity appeared to be pH dependent, but the effect was not significant. The heat of oxygenation was -11.2 kcal mol-1. At high hemoglobin concentrations, the perivisceral hemoglobin oxygen affinity was lower and the apparent pH effect and cooperativity were increased. Perivisceral and water vascular hemoglobins had spectral characteristics similar to those of other invertebrate and vertebrate hemoglobins. The perivisceral hemoglobin appeared to be electrophoretically heterogeneous and was structurally distinguishable from water vascular hemoglobin. The oxygen affinity of water vascular hemoglobin was not different from that of the perivisceral hemoglobin in spite of the difference in structure and location in the animal. The exceptionally high oxygen affinity hemoglobin of P. chilensis, a burrowing sea cucumber, may be adaptive to this animal's oxygen-limited habitat.

Developmental Changes in Ionic and Osmotic Regulation in the Dungeness Crab, Cancer magister.

The ontogeny of osmoregulation and specific ion regulation was studied in the megalopa, 1st instar juvenile, 5th instar juvenile and adult of Cancer magister. Hemolymph Na+, Cl-, K+, Mg++, and Ca++ concentrations and osmolality were measured after 8-h exposure to 100%, 75%, and 50% seawater at 10°C and 20°C. The ability to hyperosmotically regulate is present in the megalopa, and ontogenic changes occur in both ionic and osmotic regulation. First instar juvenile crabs, which are exposed to the greatest extremes of salinity and temperature in the field, are less able to osmoregulate than are the other three stages examined. Changes in Na+, Cl-, and K+ concentrations parallel total osmolality in all four stages. Hemolymph Mg++ concentrations in megalopa and juveniles acclimated to 100% seawater are more than twice that of the concentration in the adult; after 8 h in 50% seawater, the megalopa and juvenile Mg++ concentrations decrease to the level of the strongly regulated adult Mg++ concentration. Ca++ is strongly regulated by megalopas and adult crabs exposed to reduced salinity compared to the two juvenile stages. Diminished predation pressure and high food availability are proximate factors that may outweigh short-term osmoregulatory stress encountered on the tideflats during development of the juvenile crab.

Bivalve hemocyanins--a comparison with other molluscan hemocyanins.

1. The hemocyanins of the protobranch bivalves Yoldia thraciaeformis, Yoldia limatula and Acila castrensis have absorption spectra similar to other hemocyanins. 2. Hemocyanins from all three bivalves appear as six-tiered cylinders in the electron microscope (30-32 nm in diameter by 34-38 nm in height). Yoldia thraciaeformis and A. castrensis hemocyanins tend to dissociate to three-tiered half molecules with polar images and also to associate into long tubular polymers. 3. Yoldia thraciaeformis and A. castrensis hemocyanins chromatograph on Sepharose 4B gel close to gastropod hemocyanin (Mr = 9 x 10(6] rather than chiton hemocyanin (Mr = 4 x 10(6]. 4. Hemocyanins from all three vivalves have subunits with electrophoretic mobilities similar to gastropod and polyplacophoran hemocyanin subunits and slower than octopodan hemocyanin subunits. 5. These similarities between bivalve and gastropod hemocyanins are consistent with the hypothesis that bivalves and gastropods have shared a common ancestor.

Changes in myoglobin and lactate dehydrogenase in muscle tissues of a diving bird, the Pigeon Guillemot, during maturation.

1. The concentration of myoglobin (Mb) and the isozymic distribution and activity of lactate dehydrogenase (LDH) in heart and pectoralis muscle were investigated at three stages of maturation of the Pigeon Guillemot, Cepphus columba. 2. Mb is not detectable in chick pectoralis; it is present in fledgling pectoralis muscle and increases four-fold in adult pectoralis. Mb concentration in heart muscle is similar in chick and fledgling and doubles in the adult. 3. LDH activities in pectoralis muscle of fledgling and adult increase to about three times that of the chick. LDH activities in heart of chick, fledgling and adult are similar to one another. 4. All five isozymes of LDH are present in both heart and pectoralis muscle at all stages; the heart muscle shows predominantly LDH-1 isozyme, and the pectoralis, LDH-5. The relative amounts of the five isozymes in the heart extract were constant during maturation but pectoralis LDH isozymes changed during maturation towards a more even distribution of the five isozymes in the adult. 5. Changes in Mb and LDH in the Pigeon Guillemot correlate with the animal's maturation from a sedentary nest sitter to an active diver and flyer. The adult pectoralis muscle probably has both aerobic function for wing-propelled short dives and flying and anaerobic capacity for longer dives.

Thermal Vent Clam (Calyptogena magnifica) Hemoglobin.

A heterodont bivalve mollusk Calyptogena magnifica, from the East Pacific Rise and the Galápagos Rift hydrothermal vent areas, contains abundant hemoglobin in circulating erythrocytes. No other known heterodont clam contains a circulating intracellular hemoglobin. The hemoglobin is tetrameric and has a relatively high oxygen affinity, which varies only slightly between 2 degrees and 10 degrees C. The presence of hemoglobin in the clam may facilitate the transport of oxygen to be used in chemoautotrophic hydrogen sulfide metabolism.

Subunit heterogeneity of Cancer magister hemocyanin.

Hemocyanin from the Dungeness crab, Cancer magister, has been described as a 25-S two-hexamer assembly of two different 5-S subunits. We have found that at least six different 5-S polypeptide chains constitute this hemocyanin. They can be separated from one another by sodium dodecyl sulfate slab gel electrophoresis as well as by regular gel electrophoresis. The six 5-S polypeptides appear very different from one another when each SDS-treated subunit is partially digested with Staphylococcus aureus V8 protease. This pattern of six subunits is present both in hemolymph which has been examined immediately upon removal from the animal as well as in hemocyanin which has remained at room temperature for two weeks. Thus, it is unlikely that the heterogeneity is a result of proteolysis during preparation of the sample. Possible implications of the high degree of subunit heterogeneity on the protein's quaternary structure are discussed.

Marginal bands in camel erythrocytes.

The elliptical, anucleate erythrocytes of camels have been examined for the presence of marginal bands and their constituent microtubules. Lysis of erythrocytes under microtubule-stabilizing conditions readily revealed marginal bands in at least 3 % of the cells, as observed by phase-contrast and darkfield light microscopy. Microtubules plus a marginal band-encompassing network of material are visible in lysed cell whole mounts with transmission electron microscopy. Marginal band microtubules are also evident in electron micrographs of thin-sectioned camel erythrocytes identifiable as reticuloyctes on the basis of submaximal electron density (reduced haemoglobin iron content) and presence of polysomes. The results suggest that marginal bands may be involved in morphogenesis of camel erythrocytes but are not required for maintenance of their ellipticity after cells are fully differentiated.