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Impairment of cellular immunity in west Greenland sledge dogs (Canis familiaris) dietary exposed to polluted minke whale (Balaenoptera acutorostrata) blubber.

Minke whale (Balaenoptera acutorostrata) blubber is rich in organohalogen contaminants, mercury, and n-3 fatty acids. In the present study we show that a daily intake of 50-200 g of minke whale blubber causes an impairment of the nonspecific and specific cellular immune system in the West Greenland sledge dog (Canis familiaris). Immune reactions were measured by mitogen (PHA, Con A) and antigen (KLH) intradermal testing, and as the study used exposure levels similar to those of Inuits and polar bears (Ursus maritimus), it is reasonable to infer that Inuits and polar bears suffer from similar decreased resistance to diseases. It is speculated that food sources are depleted by thinning sea ice due to climate change and that more research should assess the forecasted rise in additive immunopathy effects in polar bears. Additionally, our study suggests that the fatty acid composition may be of importance when investigating combined immunotoxic effects of contaminated food resources in future Inuit and polar bear studies.

Animals↗

Heme distortions in sperm-whale carbonmonoxy myoglobin: correlations between rotational strengths and heme distortions in MD-generated structures.

We have investigated the effects of heme rotational isomerism in sperm-whale carbonmonoxymyoglobin using computational techniques. Several molecular dynamics simulations have been performed for the two rotational isomers A and B, which are related by a 180 degrees rotation around the alpha-gamma axis of the heme, of sperm-whale carbonmonoxy myoglobin in water. Both neutron diffraction and NMR structures were used as starting structures. In the absence of an experimental structure, the structure of isomer B was generated by rotating the heme in the structure of isomer A. Distortions of the heme from planarity were characterized by normal coordinate structural decomposition and by the angle of twist of the pyrrole rings from the heme plane. The heme distortions of the neutron diffraction structure were conserved in the MD trajectories, but in the NMR-based trajectories, where the heme distortions are less well defined, they differ from the original heme deformations. The protein matrix induced similar distortions on the hemes in orientations A and B. Our results suggest that the binding site prefers a particular macrocycle conformation, and a 180 degrees rotation of the heme does not significantly alter the protein's preference for this conformation. The intrinsic rotational strengths of the two Soret transitions, separated according to their polarization in the heme plane, show strong correlations with the ruffling deformation and the average twist angle of the pyrrole rings. The total rotational strength, which includes contributions from the chromophores in the protein, shows a weaker correlation with heme distortions.

Animals↗

Breeding behaviour of pilot whales revealed by DNA fingerprinting.

Most species of whale spend the majority of their lives well away from land, are capable of migrating over large distances and are difficult to identify individually. However, conservation measures require a detailed understanding of their social structure, breeding behaviour and migration patterns. The advent of DNA fingerprinting permits a systematic investigation of such parameters. In the Faeroe Islands there exists a traditional harvest of long-finned pilot whales (Globicephala melas), in which intact social groups (pods) are captured. This affords a unique opportunity to study genetic relationships within and between pods. We report here on a paternity analysis, using DNA fingerprinting, of mother-fetus pairs and males sampled from complete pods. In addition, a single, highly polymorphic minisatellite locus was used to infer degrees of relatedness between groups of fetuses and females. Taken together, our results suggest that pods consist of closely related adult females and their offspring. Sexually mature males either move frequently between pods or remain in their natal pod but refrain from mating with female relatives. Whichever hypothesis is correct, the data suggest that each male spends only a few months with the female post-mating and individual males often father several fetuses within a pod.

Animals↗

The amino acid sequence of pike-whale (lesser-rorqual) pancreatic ribonuclease.

Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.

Amino Acid Sequence↗

Structure and biological activity of finback-whale (Balaenoptera physalus L.) heparin octasaccharide. Chemical, carbon-13 nuclear-magnetic-resonance, enzymic and biological studies.

Finback-whale (Balaenoptera physalus L.) heparin was partially digested with a purified heparinase and an octasaccharide with high affinity for antithrombin III was isolated from the digest by gel filtration, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. This octasaccharide possessed high inhibitory activity for Factor Xa in the presence of antithrombin III, but was essentially inactive for thrombin-antithrombin III reaction. The anticoagulant activity determined by the activated-partial-thromboplastin-time method was very low (40-70 units/mg), although the initial whale heparin exhibited high activity (252 units/mg). On the basis of the results of chemical analyses, 13C n.m.r. spectrum and enzymic studies with purified heparinase, heparitinases 1 and 2, the predominant structure of the octasaccharide was proposed as follows: delta UA(2S) alpha 1 leads to 4GlcNS alpha 1 leads to 4IdUA alpha 1 leads to 4GlcNAc(6S) alpha 1 leads to 4GlcUA beta 1 leads to 4GlcNS(3S) alpha 1 leads to 4IdUA(2S) alpha 1 leads to 4GlcNS. Comparing this structure with those of the heparin octasaccharides so far reported, the presence of the critical structural elements for binding to antithrombin III was suggested in the pentasaccharide region situated at the reducing end of this octasaccharide. Binding to antithrombin III of the critical structural elements alone would appear to elicit the acceleration of the Factor Xa-antithrombin III reaction. Additional structural elements required for the acceleration of the thrombin-antithrombin III reaction and for the manifestation of high anticoagulant activity are discussed.

Animals↗

1H-n.m.r. and c.d. studies of haem orientational disorder in sperm-whale myoglobin and human haemoglobin.

1H-n.m.r. and c.d. studies on sperm-whale myoglobin show that the c.d. signal in the Soret region is inversely and linearly related to the proportion of minor isomer present. An alternative method, 'pH jump', is described for inducing orientational disorder in sperm-whale myoglobin without recourse to reconstitution. 1H-n.m.r. studies on human haemoglobin A indicate little heterogeneity in freshly isolated haemoglobin A, but the effect is enhanced in freeze-dried Sigma haemoglobin A.

Animals↗

Population structure of nuclear and mitochondrial DNA variation among humpback whales in the North Pacific.

The population structure of variation in a nuclear actin intron and the control region of mitochondrial DNA is described for humpback whales from eight regions in the North Pacific Ocean: central California, Baja Peninsula, nearshore Mexico (Bahia Banderas), offshore Mexico (Socorro Island), southeastern Alaska, central Alaska (Prince Williams Sound), Hawaii and Japan (Ogasawara Islands). Primary mtDNA haplotypes and intron alleles were identified using selected restriction fragment length polymorphisms of target sequences amplified by the polymerase chain reaction (PCR-RFLP). There was little evidence of heterogeneity in the frequencies of mtDNA haplotypes or actin intron alleles due to the year or sex composition of the sample. However, frequencies of four mtDNA haplotypes showed marked regional differences in their distributions (phi ST = 0.277; P < 0.001; n = 205 individuals) while the two alleles showed significant, but less marked, regional differences (phi ST = 0.033; P < 0.013; n = 400 chromosomes). An hierarchical analysis of variance in frequencies of haplotypes and alleles supported the grouping of six regions into a central and eastern stock with further partitioning of variance among regions within stocks for haplotypes but not for alleles. Based on available genetic and demographic evidence, the southeastern Alaska and central California feeding grounds were selected for additional analyses of nuclear differentiation using allelic variation at four microsatellite loci. All four loci showed significant differences in allele frequencies (overall FST = 0.043; P < 0.001; average n = 139 chromosomes per locus), indicating at least partial reproductive isolation between the two regions as well as the segregation of mtDNA lineages. Although the two feeding grounds were not panmictic for nuclear or mitochondrial loci, estimates of long-term migration rates suggested that male-mediated gene flow was several-fold greater than female gene flow. These results include and extend the range and sample size of previously published work, providing additional evidence for the significance of genetic management units within oceanic populations of humpback whales.

Alleles↗

Structure and presumptive function of the iridocorneal angle of the West Indian manatee (Trichechus manatus), short-finned pilot whale (Globicephala macrorhynchus), hippopotamus (Hippopotamus amphibius), and African elephant (Loxodonta africana).

The iridocorneal angles of prepared eyes from the West Indian manatee, short-finned pilot whale, hippopotamus and African elephant were examined and compared using light microscopy. The manatee and pilot whale demonstrated capacity for a large amount of aqueous outflow, probably as part of a system compensating for lack of ciliary musculature, and possibly also related to environmental changes associated with life at varying depths. The elephant angle displayed many characteristics of large herbivores, but was found to have relatively low capacity for aqueous outflow via both primary and secondary routes. The hippopotamus shared characteristics with both land- and water-dwelling mammals; uveoscleral aqueous outflow may be substantial as in the marine mammals, but the angular aqueous plexus was less extensive and a robust pectinate ligament was present. The angles varied greatly in size and composition among the four species, and most structures were found to be uniquely suited to the habitat of each animal.

Animals↗

Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.

Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.

Animals↗

A comprehensive and validated molecular taxonomy of beaked whales, family Ziphiidae.

DNA sequences from orthologous loci can provide universal characters for taxonomic identification. Molecular taxonomy is of particular value for groups in which distinctive morphological features are difficult to observe or compare. To assist in species identification for the little known family Ziphiidae (beaked whales), we compiled a reference database of mitochondrial DNA (mtDNA) control region (437 bp) and cytochrome b (384 bp) sequences for all 21 described species in this group. This mtDNA database is complemented by a nuclear database of actin intron sequences (925 bp) for 17 of the 21 species. All reference sequences were derived from specimens validated by diagnostic skeletal material or other documentation, and included four holotypes. Phylogenetic analyses of mtDNA sequences confirmed the genetic distinctiveness of all beaked whale species currently recognized. Both mitochondrial loci were well suited for species identification, with reference sequences for all known ziphiids forming robust species-specific clades in phylogenetic reconstructions. The majority of species were also distinguished by nuclear alleles. Phylogenetic comparison of sequence data from "test" specimens to these reference databases resulted in three major taxonomic discoveries involving animals previously misclassified from morphology. Based on our experience with this family and the order Cetacea as a whole, we suggest that a molecular taxonomy should consider the following components: comprehensiveness, validation, locus sensitivity, genetic distinctiveness and exclusivity, concordance, and universal accessibility and curation.

Actins↗

G-banded karyotype and ideogram for the North Atlantic right whale (Eubalaena glacialis).

Published cytogenetic data for extant cetacean species remain incomplete. In a review of the literature, we found karyotypic information for 6 of the 13 tentatively recognized species of the suborder Mysticeti (baleen whales). Among those yet to be described is the critically endangered North Atlantic right whale (Eubalaena glacialis). Herein, we describe and propose a first-generation G-banded karyotype and ideogram for this species (2n = 42), obtained from peripheral blood chromosome preparations from a stranded male calf. This information may prove useful for future genetic mapping projects and for interspecific and intraspecific genomic comparisons by techniques such as zoo-FISH.

Animals↗

The 2-electron reduction of sperm whale ferryl myoglobin by ethanol.

Upon the addition of hydrogen peroxide or ethyl hydroperoxide to sperm whale metmyoglobin (MbIII) in the presence of ethanol, MbIII was converted to oxymyoglobin (MbIIO2) under aerobic conditions and carboxymyoglobin (MbIICO) under CO-saturated conditions. MbIIO2 and MbIICO were also formed when ethanol was added to ferryl myoglobin (MbIV) which had been formed from the reaction of MbIII with hydrogen peroxide. From the stoichiometry, the primary reaction was formulated as follows. MbIV + ethanol----MbII + acetaldehyde The reaction was optimal at pH 7.0-7.5. Sperm whale ferryl myoglobin was reduced less effectively by methanol and n-butanol, but not at all by sec- and tert-butanols. The reduction of ferryl hemoproteins by ethanol was slower with horse heart myoglobin and was not observed with bovine hemoglobin or horseradish peroxidase.

Animals↗

Contribution of chemical 6-O-sulfation of the aminodeoxyhexose residues in whale heparin with high affinity for antithrombin III to its anticoagulant properties.

The tributylammonium salt of whale (Balaenoptera borealis L.) intestinal heparin with high affinity for antithrombin III, whose degrees of sulfate-substitution in D-glucosamine and L-iduronic acid residues are GlcNS 0.738, GlcN6S 0.384, and IdoA2S 0.510 mol, was reacted with 2.5, 5.0, or 10.0 mol of pyridine-sulfur trioxide/mol of available hydroxyl groups in N,N-dimethylformamide at -10 degrees C for 1 h. Both chemical and NMR spectroscopic analyses revealed that an exclusive 6-O-sulfation of the D-glucosamine residues proceeded, according to the amount of the sulfating reagent used (GlcN6S: 0.476, 0.585, and 0.641 mol, respectively), the degree of sulfation at other natural substitution positions in the polysaccharide being unchanged, without any detectable unnatural sulfate-substitution. Biological examination of these products indicated that the 6-O-sulfation in the original whale heparin resulted in significant increases in blood clotting and anti-Factor IIa activities (maximal 43 and 82% increases, respectively), and in a moderate increase in the ability to bind antithrombin III, that is, in anti-Factor Xa activity and in intrinsic fluorescence enhancement of the protein (maximal 28 and 30% increases, respectively), together with a maximal 10% increase in the proportion of heparin species with higher affinity for antithrombin III, released with 1.0-3.0 M NaCl from antithrombin III-Sepharose.

Animals↗

Isolation and partial characterization of growth hormone from fin whale pituitary glands.

A highly purified somatotropin (growth hormone) has been isolated from fin whale (Balaenoptera physalus) pituitary glands in a yield of 1,014 mg/kg of wet weight tissue. The preparation was shown to be homogenous by isoelectric focusing on polyacrylamide with growth-promoting potency comparable to that of ovine growth hormone by the rat tibia test. The molecular weight was found to be 20,000 daltons by SDS gel electrophoresis and 40,000 daltons by exclusion chromatography on Sephadex G-100 at pH 8.2. The amino acid composition and alpha-helix content of fin whale somatotropin were similar to those of human, bovine, and ovine hormones. The circular dichroism in the side chain region, however, differed from those of the mammalian hormones. The NH2-terminal amino acid sequence was nearly identical with those of the bovine and ovine hormones as determined by the fluorescein isothiocyanate method.

Amino Acids↗

Comparative studies on the structures of highly active and relatively inactive forms of whale heparin.

Whale heparin was separated by affinity chromatography on an antithrombin III-Sepharose column into two distinct fractions. The high-affinity fraction accounted for most the anticoagulant activity of the unfractionated material, while the low-affinity fraction was relatively inactive. The yields of the two fractions were substantially equivalent. No significant difference was observed between these fractions in terms of electrophoretic mobilities on cellulose acetate membrane and analytical data except for the contents of N-acetylglucosamine and N-sulfoglucosamine. The highly active form contained more N-acetylglucosamine and less N-sulfoglucosamine than the relatively inactive form. The two fractions were separately subjected to the sequential digestion with purified heparinase and heparitinase, and the oligosaccharide fractions were isolated from the digests by DEAE-cellulose column chromatography, followed by preparative paper chromatography. The purified compounds were then characterized by routine chemical and physical methods. Compound 1, delta 4,5hexosyuronic acid1 leads to 4N-acetylglucosamine, was exclusively obtained from the highly active form, whereas compound 3a, delta 4,5hexosyluronic acid1 leads to 4N-acetylglucosamine 6-sulfate, and compound 3b delta 4,5hexosyluronic acid1 leads to 4-sulfoglucosamine, were the only ones obtained from the relatively inactive form. The yields of other oligosaccharide fractions from both forms were comparable. The present data suggest that an N-acetylglucosamine-containing oligosaccharide structure in whale heparin is essential for binding to antithrombin II.

Animals↗

Patterns of genetic variability at individual minisatellite loci in minke whale Balaenoptera acutorostrata populations from three different oceans.

The genetic variability at six cloned minisatellite loci was analyzed in minke whale populations from the North Atlantic, North Pacific, and Antarctic Oceans. Three loci displayed only a few different alleles in each of the three populations, with heterozygosity ranging from 0.00 to 0.47, and three loci revealed many different alleles in at least two fo the three populations, with heterozygosity ranging up to 0.98. Using small sample sizes, samples from two adjacent Antarctic Management Areas were not found to differ significantly in allele frequencies at any of the six loci. The use of principal coordinate analysis to detect multilocus disequilibria was explored. No significant evidence was found of intrapopulation heterogeneity within the pooled Antarctic sample. Pronounced interoceanic differences were observed at every locus, confirming the existence of genetic isolation found earlier using more conventional marker systems. The populations from the three oceans appear to have diverged to such a degree that the hypervariable loci have had time to evolve independently and arrive at different evolutionary stages in different populations. The frequency of undetected "null" alleles is remarkably high in minke whale populations compared to human populations and is probably a result of the cloning protocol used. Minisatellite loci are shown to provide a powerful population genetic tool, supplying levels of resolution appropriate to different degrees of evolutionary divergence.

Alleles↗

Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas)

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.

Alleles↗

Characteristics of a conserved 1,579-bp highly repetitive component in the killer whale, Orcinus orca.

A tandemly organized, highly repetitive DNA component of the killer whale was sequenced. The length of the repeat was 1,579 bp. This unit, which characterizes all delphinids, shows stringent hybridization homology with a 1,740-bp repeat that is characteristic of all other cetacean families. The 1,579-bp component comprises approximately 15% of the killer-whale genome, in which it is repeated 4-5 X 10(5) times. Computer analysis of the sequence showed no linear repetition within the component. This indicates that the 1,579-bp unit has not evolved by amplification of shorter repeats. Several inverted repeats of substantial length were found in the 1,579-bp unit. The most conspicuous of these was a 72-bp sequence that deviated from matching in only three positions. The 72-bp sequence occurs within an open reading frame 330 bp in length. Transcriptional activity was registered in the cloned repeat in a cell-free system. The length of the transcript was approximately 340 nucleotides. The chromosomal localization of the 1,579-bp repeat was determined by in situ hybridization. The repeat was present in eight of 21 autosomal pairs and was found in almost all C-band-positive (constitutive heterochromatin) regions of the karyotype.

Animals↗