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Physiological colour change was investigated in the blackspotted rockskipper Entomacrodus striatus in Moorea, French Polynesia. Fish colour cycled with significant autocorrelation over the 30 min observation period and was not affected by observation temperature (27 and 31° C). Cycling depended most on dark and yellow pigments (as assayed by separation of colours via software), and therefore, it was hypothesized that short-term cycling was driven by melanophores and xanthophores. [ABSTRACT FROM AUTHOR]

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. In this study, we examined expression of zebrafish cadherin-2 (Cdh2, N-cadherin) and cadherin-4 (Cdh4, R-cadherin) in the visual system of adult zebrafish after eye or optic nerve lesions using immunocytochemistry and immunoblotting. Both Cdh2 and Cdh4 immunoreactivities were specifically up-regulated in regenerating retina and/or the optic pathway. Furthermore, temporal expression patterns of these two cadherins were distinct during the regeneration of the injured tissues. Cadherins have been shown to regulate axonal outgrowth in the developing nervous system, but this is the first report, to our knowledge, of increased cadherin expression associated with axonal regeneration in the vertebrate central nervous system. Our results suggest that both Cdh2 and Cdh4 may be important for regeneration of injured retinal ganglion cell axons. [Copyright &y& Elsevier]

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.

Tissues from bony fish were screened with anti-mouse leptin antibodies to detect the presence of the fat-regulating hormone in fishes. Low molecular-weight (16 kDa) immunoreactive bands were detected in blood, brain, heart and liver of green sunfish (Lepomis cyanellus), bluegill sunfish (Lepomis macrochirus), largemouth bass (Micropterus salmoides), white crappie (Pomonix annularis), channel catfish (Ictalurus punctatus), and rainbow trout (Oncorhynchus mykiss). To further verify that we had identified leptin, the response of fish "leptin" was measured in fed and fasted green sunfish. Fed sunfish had approximately threefold higher concentration of leptin in blood than did fasted sunfish (fed vs. fasted; 0.599 +/- 0.03 microg/microl vs. 0.196 +/- 0.04 microg/microl; P > F = 0.0001), which is consistent with mammalian models of leptin function. Brain leptin concentration is also positively correlated with percent body fat in white crappie and bluegill. Based upon electrophoretic mobility, immunoreactivity, response to fasting, and correlation with adiposity, we believe we have the first evidence for leptin expression in an ectotherm.

Leptin is a hormone that regulates energy expenditure and body mass in mammals, and it has attracted considerable attention because of its potential in treating human obesity. Comprehensive data from both pathological and non-pathological systems strongly support a role for leptin in regulating energy metabolism, in thermoregulation and in regulating the onset of puberty. We report here that daily injections of recombinant murine leptin in fence lizards (Sceloporus undulatus) produce phenotypic effects similar to those observed when leptin injections are given to mice. Lizards injected with leptin had body temperatures 0.6 degrees C higher, ate 30 % less food and showed a 14 % reduction in activity rates, and females showed a 2. 5-fold increase in resting metabolic rates, compared with lizards injected with vehicle only (phosphate-buffered saline). We also detected native lizard leptin using an immunoassay. Our results indicate that leptin is expressed in ectotherms and may be conserved both functionally and structurally. In the wake of unprecedented research activity on the role of leptin as a cause of, and potential treatment for, human obesity, we believe that other applications of leptin research have been ignored. For example, the response of lizards to leptin injection in our study has important implications for two broad areas of research in evolutionary biology: the evolution of age at first reproduction and of endothermy. We argue that research in these areas, previously limited to comparative approaches, may now benefit from experimental manipulations using leptin.

A variety of circulating fuels can support the work of the teleost gill. Previous work indicates, however, that unlike other aerobic tissues from teleosts, the gill may have a limited capacity to oxidize fatty fuels. We determined capacities for catabolism of carbohydrate, fatty acids, and amino acids in four species of temperate marine or euryhaline teleosts representing distinct lineages. In addition, we assessed the capacity for fatty acid oxidation in the gill from an Antarctic species. Activities of rate-limiting or regulatory enzymes from pathways of energy metabolism were measured at physiological temperatures (15 degrees or 1 degrees C). In the temperate species, ATP yields from glucose are 3- to 30-fold greater (varying with species) than ATP yields from a monounsaturated fatty acid, while ATP generation from glutamate is 2-50 times greater than similar capacities for the lipid fuel. Like the temperate species, capacity for beta-oxidation of fatty acids is limited in the Antarctic species. A positive linear correlation between activities of citrate synthase (central pathway of oxidative metabolism) and hexokinase (glycolysis) adds further support to the hypothesis that glucose is a preferred metabolic fuel in gill. Our results also demonstrate that fatty acid-binding protein is present in the gill of teleost fishes. It is likely that this protein plays a more important role facilitating anabolic pathways in lipid metabolism rather than fatty acid oxidation in the gill of teleost fishes., (Copyright 1999 Wiley-Liss, Inc.)

We have cloned a group of cDNAs that encodes the skeletal ryanodine receptor isoform (RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs encode a protein of 5,081 amino acids with a calculated molecular mass of 576,302 Da. The deduced amino acid sequence shows strong sequence identity to previously characterized RyR1 isoforms. An RNA probe derived from a clone of the full-length marlin RyR1 isoform hybridizes to RNA preparations from extraocular muscle and slow-twitch skeletal muscle but not to RNA preparations from fast-twitch skeletal or cardiac muscle. We have also isolated a partial RyR clone from marlin and toadfish fast-twitch muscles that shares 80% sequence identity with the corresponding region of the full-length RyR1 isoform, and a RNA probe derived from this clone hybridizes to RNA preparations from fast-twitch muscle but not to slow-twitch muscle preparations. Western blot analysis of slow-twitch muscles in fish indicates the presence of only a single high-molecular-mass RyR protein corresponding to RyR1. [3H]ryanodine binding assays revealed the fish slow-twitch muscle RyR1 had a greater sensitivity for Ca2+ than the fast-twitch muscle RyR1. The results indicate that, in fish muscle, fiber type-specific RyR1 isoforms are expressed and the two proteins are physiologically distinct.

This report provides the first evidence for the existence of two distinct types of fatty acid-binding protein (FABP) in cardiac tissue of vertebrates. Four species of Antarctic teleost fish (Chaenocephalus aceratus, Cryodraco antarcticus, Gobionotothen gibberifrons and Notothenia coriiceps) exhibited two FABP mRNAs of 1. 0 kb and 0.8 kb, which we have termed Hh-FABP and Had-FABP (isolated from Heart tissue, with similarity to mammalian heart-type FABP or mammalian adipose-type FABP respectively). These FABP types appear to be products of distinct genes. Both FABP transcripts were abundant in cardiac and aerobic pectoral muscle. However, relative abundance of the two types varied distinctly among other tissues such as kidney, brain, spleen and white muscle. Neither FABP type was expressed in liver or intestine. The coding regions of Hh-FABP and Had-FABP cDNAs from the same species are only approximately 60% identical with one another. However, homologues of each FABP species, which exhibit >98% identity to their respective types, were isolated from three other Antarctic teleosts. Phylogenetic analysis of aligned amino-acid sequences places Hh-FABP with other vertebrate heart-type FABPs, and Had with adipose/cutaneous FABPs. Expression of two distinct FABPs in cardiac tissue of Antarctic teleosts may be related to their ability to both utilize fatty acid as the primary metabolic fuel and to store lipid intracellularly.

The important intracellular oxygen-binding protein, myoglobin (Mb), is thought to be absent from oxidative muscle tissues of the family of hemoglobinless Antarctic icefishes, Channichthyidae. Within this family of fishes, which is endemic to the Southern Ocean surrounding Antarctica, there exist 15 known species and 11 genera. To date, we have examined eight species of icefish (representing seven genera) using immunoblot analyses. Results indicate that Mb is present in heart ventricles from five of these species of icefish. Mb is absent from heart auricle and oxidative skeletal muscle of all species. We have identified a 0.9-kb mRNA in Mb-expressing species that hybridizes with a Mb cDNA probe from the closely related red-blooded Antarctic nototheniid fish, Notothenia coriiceps. In confirmation that the 0.9-kb mRNA encodes Mb, we report the full-length Mb cDNA sequence of the ocellated icefish, Chionodraco rastrospinosus. Of the eight icefish species examined, three lack Mb polypeptide in heart ventricle, although one of these expresses the Mb mRNA. All species of icefish retain the Mb gene in their genomic DNA. Based on phylogeny of the icefishes, loss of Mb expression has occurred independently at least three times and by at least two distinct molecular mechanisms during speciation of the family.

Binding affinity and binding-pocket polarity is determined for intracellular fatty acid-binding protein (FABP) from aerobic muscle of Chaenocephalus aceratus, the Antarctic icefish, and from rat heart. FABPs bind fatty acids via weak-bond forces (both ionic and hydrophobic), and these bond forces are temperature sensitive, yet FABPs are present in animals whose body temperatures range over nearly 40 degrees C. To investigate FABP's sensitivity to body temperature, fatty acid binding affinity (Kd) was determined for both rat heart-FABP and icefish heart-FABP at two physiological temperatures (0 degrees C or 37 degrees C). Saturated and unsaturated fatty acids (16:0 and 16:1), delivered in model membranes (liposomes) whose composition is typical of either Antarctic fish (16:0/22:6 phosphatidylcholine) or mammals (bovine-heart phosphatidylcholine) were examined. Incubation at 0 degree C or 37 degrees C dose not significantly affect Kd for rat heart FABP, regardless of liposome composition or fatty acid ligand (Kd = 0.686 +/- 0.127 - 1.129 +/- 0.356 microM at 0 degree C, 0.775 +/- 0.307 - 1.605 +/- 0.427 microM at 37 degrees C). Incubation temperature significantly affects icefish FABP's affinity for 16:1 (0.626 +/- 0.093 microM at 37 degrees C vs. 1.896 +/- 0.343 microM at 0 degree C for fatty acid presented in Antarctic fish liposomes; 0.331 +/- 0.101 microM at 37 degrees C vs. 0.949 +/- 0.121 microM at 0 degree C for bovine heart liposomes) but not 16:0. Kd is not significantly different between FABPs under any set of conditions (with one exception: Kd is significantly lower in rat FABP vs. icefish FABP for 16:0 at 0 degree C for fatty acids delivered in bovine heart liposomes). Although Kd values are largely equivalent between the two FABPs, relative contributions from ionic vs. hydrophobic weak-bond forces are different between the two animals. Rat heart FABP has a binding pocket that is significantly more nonpolar than that of icefish FABP (as measured by quantum yield of the bound fluorescent fatty-acid analogue (PA-DPH); Q = 0.067 +/- 0.008 vs. 0.034 +/- 0.005 at 0 degree C, 0.030 +/- 0.003 vs. 0.019 +/- 0.002 at 37 degrees C). This suggests that rat-heart FABP realizes a micromolar Kd with a greater reliance upon hydrophobic interactions than does icefish FABP.

Intracellular fatty acid-binding proteins (FABPs) are small (15 kDa), highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. Fifteen isoforms have been identified and characterized to date. Members of the FABP protein family share many common characteristics: they have a common "clamshell" tertiary structure despite significant variation in primary structure; they bind their ligands with 1:1 molar stoichiometry and micromolar affinity (with the exception of liver type FABP, which binds two fatty acids per FABP); they are ubiquitously expressed, yet are found at greatest concentration in tissues with high capacities to oxidize or store lipids; and their level of expression changes with conditions that affect overall lipid metabolism. Because of the ligands bound by FABP and FABP's tissue distribution, it is thought that FABPs play a role in the intracellular metabolism of lipids; however, a precise function(s) for FABP has not been defined. This review highlights contributions from researchers studying FABP in nonmammalian systems. FABPs isolated from birds, fish, amphibians, insects, and flatworms have similar structures to mammals in FABPs, yet they are expressed in systems with unique lipid metabolisms. Because mammals are closely related and have relatively invariant fatty acid metabolisms, non-mammals provide alternative systems that may reveal the elusive function(s) of this protein family.

Intracellular fatty acid-binding protein is purified and characterized from aerobic skeletal muscle of the Antarctic icefish Chaenocephalus aceratus. Molecular mass of C. aceratus FABP (CA-FABP) is 14,936 Da as estimated by electrospray mass spectrometry. CA-FABP is expressed at an intracellular concentration of 0.984 +/- 0.115 mg CA-FABP g-1 wet weight aerobic muscle and binds 0.859 +/- 0.013 moles oleic acid per mole of protein at a physiological temperature of 0 degrees C. Dissociation constants (KdS for various fatty acid ligands range from 1.38 to 2.71 microM; KdS are not significantly different among palmitic acid (16:0), palmitoleic acid (16:1), and oleic acid (18:1). Competition assays reveal that CA-FABP does not have preferential affinity for the very-long-chain, polyunsaturated fatty acids that are common in Antarctic fish (e.g., docosahexaenoic acid; 22:6). Partial amino acid sequence from CA-FABP aligns with mammalian heart-type FABPs with as high as 74% identity. These data are strikingly similar to mammalian values, yet they are derived from an organism that is distant from mammals in terms of phylogeny, body temperature, and physiology. This suggests that the FABP family is conserved not only in primary sequence, but also in its physiological properties.

Mitochondrial volume density (Vv [mit]) distributions were measured with a test pattern of concentric rings centered upon randomly chosen capillaries in oxidative skeletal muscle cells of two Antarctic fishes, Trematomus newnesi and Notothenia gibberifrons. Vv(mit) in both species was highest in the ring closest to the capillary, minimal further from the capillary (at a distance that was characteristic for each species), and rose in the annuli furthest from the capillary. Plots of Vv(mit) against total area between each ring and the central capillary fit the form of a second-order polynomial (r2 greater than 0.9). If PO2 or blood-borne metabolite concentration predicates the pattern of Vv(mit) distribution, minimal Vv(mit) is at the same position as the minimum in concentration or gaseous partial pressure of capillary-supplied commodities. This minimum is the boundary between cylinders of tissue being supplied by adjacent capillaries, and thus delineates the maximal diffusion-distance for capillary-supplied commodities. Maximal diffusion-distance (microns) for T. newnesi = 26.23 +/- 1.64; N. gibberifrons = 21.45 +/- 0.51. For O2, maximal diffusion-distance conventionally is referred to as Krogh's radius, R. With an easily obtained estimate of numerical capillary density, these R values can be used to calculate a capillary tortuosity constant (c[k,0]) and capillary length density (Jv[c,f]). c(k,0) values were also determined using an established method, and R and Jv(c,f) values calculated from these values did not significantly differ from values determined from mitochondrial distributions. Mitochondrial distribution analysis may more accurately reflect changes in capillary blood flow and heterogeneity of diffusion and solubility constants within muscle than currently existing techniques. Similar distributions of Vv(mit) reported for several species of vertebrates suggest wide applicability of the method.