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PubMed1999

Scaling of the cetacean middle ear.

Functionally interesting dimensions of the tympano-periotic complex were measured and compared in 18 odontocete and six mysticete species, ranging from small porpoises to the blue whale. We determined (i) the masses of the tympanic and periotic bones (T and P) and of the ossicles malleus, incus, and stapes (M, I and S), (ii) the volume occupied bythe tympanic bone (V), (iii) the areas of the tympanic plate and oval window (A1 and A2), (iv) the thickness of the tympanic plate (D), and (v) the densities of the ossicles (dM, dI, and dS). In most cases, roughly isometric scaling was found in both toothed and baleen whales. P is isometric to T, and the tympanic bone is structurally isometric in all species studied, although not within mysticetes as a group, shown by the isometric relations of V to T, of T(2/3) to A1, and of D to square root(A1). The essentially isometric scaling of the tympanic bone provides a basis for the functional models described by Hemilä et al. (1999). The relation of S to M+I is also isometric, but the relation of M+I+S to T is negatively allometric, as is the relation of A2 to A1, both with slopes close to 2/3. The possible functional implication of this allometry is unknown. The mean ossicular density is 2.64 g/cm3 for odontocetes, and 2.35 g/cm3 for mysticetes. The highly mineralized and convex tympanic plate provides cetaceans with a uniquely large and stiff sound collecting area.

Animals↗
PubMed1999

Anatomy of the cranial nerve foramina in the Risso's dolphin (Grampus griseus).

In the present study, we examined the cranial nerve foramina of Risso's dolphin (Grampus griseus). There were two distinguishable characters in the cranial nerve foramina compared with terrestrial mammals. One was that the foramen infraorbitale was composed of several holes, but not a single hole. They should therefore be termed foramina infraorbitales. The infraorbital nerves ran through these foramina, went into the 'melon' and then branched in a complicated fashion. The facial nerve innervated the muscles surrounding the melon. A well-developed infraorbital and facial nerve complex may control the melon. Another was the presence of a porus acusticus internus and independent tympano-periotic bone. The separate ear bone forced the vestibulocochlear and facial nerves to exit from the cranial cavity through the porus acusticus internus. The independent ear bone structure may shut off the noise from the cranial bone to the periotic bone with a true receptor of hearing. It may be an adaptation for an acute sense of hearing. Compared with other dolphins, the cranial foramina of Risso's dolphin are definitely separate. The structure of the foramina is similar to that of pilot whales, but not to dolphins, so that Grampus may be closely allied to pilot whales.

Animals↗
PubMed2000

Body size and skeletal muscle myoglobin of cetaceans: adaptations for maximizing dive duration.

Cetaceans exhibit an exceptionally wide range of body mass that influence both the capacities for oxygen storage and utilization; the balance of these factors is important for defining dive limits. Furthermore, myoglobin content is a key oxygen store in the muscle as it is many times higher in marine mammals than terrestrial mammals. Yet little consideration has been given to the effects of myoglobin content or body mass on cetacean dive capacity. To determine the importance of myoglobin content and body mass on cetacean diving performance, we measured myoglobin content of the longissimus dorsi for ten odontocete (toothed whales) and one mysticete (baleen whales) species ranging in body mass from 70 to 80000 kg. The results showed that myoglobin content in cetaceans ranged from 1.81 to 5.78 g (100 g wet muscle)(-1). Myoglobin content and body mass were both positively and significantly correlated to maximum dive duration in odontocetes; this differed from the relationship for mysticetes. Overall, the combined effects of body mass and myoglobin content accounts for 50% of the variation in cetacean diving performance. While independent analysis of the odontocetes showed that body mass and myoglobin content accounts for 83% of the variation in odontocete dive capacity.

Adaptation, Physiological↗
PubMed2001

Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus.

A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale. These strains were identified as brucellae by conventional typing tests. However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called Brucella maris. In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species. This observation was also recently made for a minke whale Brucella isolate. The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals. By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates. Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals. Therefore, grouping the marine mammal Brucella isolates into one species Brucella maris seems inappropriate unless the currently recognized Brucella species are grouped. With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B. pinnipediae and B. cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus.

Animals↗
PubMed2006

Distribution of Pholeter gastrophilus (Digenea) within the stomach of four odontocete species: the role of the diet and digestive physiology of hosts.

We compared the distribution of the digenean Pholeter gastrophilus in the stomach of 27 harbour porpoises, Phocoena phocoena, 27 striped dolphins, Stenella coeruleoalba, 18 bottlenose dolphins, Tursiops truncatus, and 100 long-finned pilot whales, Globicephala melas. The stomach of these species is composed of 4 chambers of different size, structure and function. In all species, P. gastrophilus was largely restricted to the glandular region of the stomach, but the parasite tended to favour the fundic chamber in bottlenose dolphins and harbour porpoises, the pyloric chamber in pilot whales, and none in striped dolphins. However, predictability at infrapopulation level was generally low, suggesting a weak preference of P. gastrophilus for any of the chambers. Three hypotheses were tested to investigate a common cause for the distribution of P. gastrophilus in all host species, namely, colonization of chambers was (1) sequential, (2) dependent on chamber size, or (3) dependent on the passage time of food through the whole stomach. The latter hypothesis was indirectly tested by assuming, based on previous evidence from other vertebrates, that the greater the size of the stomach and/or the energy content of prey, the greater the delay of food passage. We found no compelling evidence that chamber colonization was sequential, or related to chamber size in any species. However, the distribution of P. gastrophilus was significantly more anteriad when the host species had larger stomachs and, particularly, when hosts fed on prey with higher caloric content. Accordingly, the stomach distribution of P. gastrophilus at this scale seems to be passively driven by features of the diet and digestive physiology of each host species. This study provides a general framework to formulate null hypotheses in future studies on microhabitat choice by parasites.

Animals↗
PubMed1992

High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.

Animals↗
PubMed1994

Electron-nuclear coupling to the proximal histidine in oxy cobalt-substituted distal histidine mutants of human myoglobin.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate electron-nuclear coupling to the N epsilon of the proximal histidine (F8, His93) imidazole in oxyCo(II)-substituted distal histidine (E7, His64) mutants (His-->Leu, His-->Val, His-->Gly, His-->Gln) and recombinant wild-type human myoglobins (Mbs). Nuclear hyperfine and nuclear quadrupole coupling constants decrease in the order: H64L > H64V > or = H64G approximately H64Q > wild-type. The differences in couplings found for the four mutant proteins are correlated with the differences in polarity of the E7 side chain. On the basis of the relative orientation of the nuclear quadrupole and g tensors, obtained by computer simulation of ESEEM spectra, the Co-O-O bond angle of H64G and H64Q appears to be similar to that of oxyCo sperm whale Mb (and possibly wild-type human Mb) at room temperature [Hori et al. (1982) J. Biol. Chem. 257, 3636], while that in H64V and H64L is more obtuse. ESEEM measurements in D2O demonstrate the presence of a hydrogen bond between the distal histidine and bound O2 in the wild-type protein, as was found in oxyCo sperm whale and horse Mbs [Lee et al. (1992) Biochemistry 31, 7274]. This hydrogen bond leads to a reduction in the N epsilon coupling in the wild-type protein as compared to that in the E7 mutants. No hyperfine-coupled deuterons were found in any of the mutants, and therefore, the proposed hydrogen bond between bound O2 and the distal glutamine in H64Q [Ikeda-Saito et al. (1991) J. Biol. Chem. 266, 23641] could not be substantiated.

Chemical Phenomena↗
PubMed1994

Conformational stability of heme proteins in vacuo.

The function and conformational stability of myoglobin were studied in vacuo by using an electrospray mass spectrometer. The electrospray technique gently transfers protein molecules from the solution phase to the gas phase, and solvent-free protein ions are produced in the mass spectrometer. Horse myoglobin was dissolved at neutral pH, Fe3+ in the heme was reduced to Fe2+ to produce the biologically active oxymyoglobin in solution, and then the protein was isolated in vacuo. A molecular ion (17,601 Da) corresponding to the molecular mass of oxymyoglobin (17,600.0 Da) was observed in the mass spectrum. This demonstrates that the protein retains a heme and an oxygen molecule in the gas phase. Since the biological function of myoglobin is to carry an oxygen molecule, this is the first observation that a protein is functional in the absence of solvent. Gas-phase "unfolding" of myoglobin was also studied. Collisions of accelerated protein ions with nitrogen curtain gas at a quadrupole guidance lens or argon gas introduced at a second quadrupole increase the "molecular temperature" of myoglobin, resulting in release of the heme from myoglobin. Apomyoglobin produced at the quadrupole guidance lens showed a larger collisional cross section than that of myoglobin, revealing conformational disordering of the protein. The gas-phase unfolding of horse and whale myoglobins and the alpha-chain of human hemoglobin induced at the second quadrupole were studied as a function of the argon gas thickness. Horse and whale myoglobins showed the same gas-phase stability, whereas the alpha-chain of human hemoglobin was less stable.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species.

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.

Carbon Isotopes↗
PubMed1988

Anomalously slow cyanide binding to Glycera dibranchiata monomer methemoglobin component II: implication for the equilibrium constant.

In comparison to sperm whale metmyoglobin, metleghemoglobin a, methemoglobins, and heme peroxidases, the purified Glycera dibranchiata monomer methemoglobin component II exhibits anomalously slow cyanide ligation kinetics. For the component II monomer methemoglobin this reaction has been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0, employing 100-250-fold mole excesses of potassium cyanide at each pH. At 20 degrees C, with micromolar protein concentrations, kobsd varies between 9.11 x 10(-5) s-1 at pH 6.0, 100-fold KCN mole excess, and 1.12 x 10(-2) s-1 at pH 9.0, 250-fold KCN mole excess. Our analysis shows that the concentration-independent bimolecular rate constant (k1app) is small in comparison to those of the other heme proteins. For example, at pH 7.0 it is 0.491 M-1 s-1, compared to 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase; 111 M-1 s-1 for guinea pig methemoglobin; approximately 400 M-1 s-1 for sperm whale metmyoglobin; and 692 M-1 s-1 for soybean metleghemoglobin a, at the same pH and similar temperatures. Furthermore, our results show that the dissociation rate is extremely slow, with k-1app no larger than 10(-6) s-1. Separation of the bimolecular rate constant into contributions from kCN- (the rate constant for CN- binding) and from kHCN (the rate constant for HCN binding) shows that the former is approximately 90 times greater. These results indicate that cyanide ligation reactions are not instantaneous for this protein, which is important for those attempting to study the ligand-binding equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin.

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].

Animals↗
PubMed1990

Rearrangement of the distal pocket accompanying E7 His----Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket.

Two-dimensional 1H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO2). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C zeta H proton of the Phe CD4 was found to move toward the iron of the heme by approximately 4 A relative to the position of sperm whale MbCO, requiring minimally a 3-A movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His----Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.

Animals↗
PubMed2002

Perfluorooctanesulfonate and related fluorinated hydrocarbons in marine mammals, fishes, and birds from coasts of the Baltic and the Mediterranean Seas.

Perfluorooctanesulfonate (PFOS; C8F17SO3-), perfluorooctanesulfonamide (FOSA; C8F17SO2NH2), perfluorohexanesulfonate (PFHxS; C6F13SO3-), and perfluorooctanoate (PFOA; C7F15CO2-) were detected in 175 samples of liver and blood of bluefin tuna (Thunnus thynnus), swordfish (Xiphias gladius), common cormorants (Phalacrocorax carbo), bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), common dolphins (Delphinus delphi), fin whales (Balenoptera physalus), and long-finned pilot whales (Globicephala melas) from the Italian coast of the Mediterranean Sea and in livers of ringed seals (Phoca hispida), gray seals (Halichoerus grypus), white-tailed sea eagles (Haliaeetus albicilla), and Atlantic salmon (Salmo salar) from coastal areas of the Baltic Sea. PFOS was detected in all of the wildlife species analyzed. Concentrations of PFOS in blood decreased in order of bottlenose dolphins > bluefin tuna > swordfish. Mean PFOS concentrations (61 ng/ g, wet wt) in cormorant livers collected from Sardinia Island in the Mediterranean Sea were less than the concentrations of PFOA (95 ng/g, wetwt). PFOS concentrations in cormorant livers were significantly correlated with those of PFOA. FOSA was found in 14 of 19 livers or blood samples of marine mammals from the Mediterranean Sea. The highest concentration of 878 ng FOSA/g, wet wt, was found in the liver of a common dolphin. Livers of ringed and gray seals from the Bothnian Bay in the Baltic Sea contained PFOS concentrations ranging from 130 to 1,100 ng/g, wet wt. No relationships between PFOS concentrations and ages of ringed or gray seals were observed. Concentrations of PFOS in livers of seals were 5.5-fold greater than those in corresponding blood. A significant positive correlation existed between the PFOS concentrations in liver and blood, which indicates that blood can be used for nonlethal monitoring of PFOS. Trend analysis of PFOS concentrations in livers of white-tailed sea eagles collected from eastern Germany and Poland since 1979 indicated an increase in concentrations during the 1990s. Livers of Atlantic salmons did not contain quantifiable concentrations of any of the fluorochemicals monitored. PFOS is a widespread contaminant in wildlife from the Baltic and the Mediterranean Seas, while FOSA and PFOA were detected only in certain locations indicating their sporadic spatial distribution.

Alkanesulfonic Acids↗
PubMed2005

Investigations of the potential influence of environmental contaminants on the thymus and spleen of harbor porpoises (Phocoena phocoena).

Harbor porpoises from the German North and Baltic Seas exhibit a higher incidence of bacterial infections compared to whales from less polluted arctic waters. The potential adverse effect of environmental contaminants such as polychlorinated biphenyls (PCBs) and heavy metals on the immune system and the health status of marine mammals is still discussed controversially. The aim of the present study was to investigate the possible influence of PCB, polybrominated diphenyl ether (PBDE), toxaphene, (p,p'-dichlorodiphenyl)trichlorethane (DDT), and (p,p'-dichlorodiphenyl)dichlorethene (DDE) on the immune system of harbor porpoises. Lymphoid organs are influenced by a variety of factors, and therefore special emphasis was given to separating the confounding effect of age, health status, nutritional state, geographical location, and sex from the effect of contaminant levels upon thymus and spleen. Contaminant analysis and detailed pathological examinations were conducted on 61 by-caught and stranded whales from the North and Baltic Seas and Icelandic and Norwegian waters. Stranded harbor porpoises were more severely diseased than by-caught animals. Thymic atrophy and splenic depletion were significantly correlated to increased PCB and PBDE levels. However, lymphoid depletion was also associated with emaciation and an impaired health status. The present report supports the hypothesis of a contaminant-induced immunosuppression, possibly contributing to disease susceptibility in harbor porpoises. However, further studies are needed to determine if lymphoid depletion is primarily contaminant-induced or secondary to disease and emaciation in this cetacean species.

Animals↗
PubMed2003

Conservation of the 3'-untranslated regions of calmodulin mRNAs in cetaceans.

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5'- and 3'-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3'- untranslated region (3'UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3'UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3'UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3'UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3'UTR sequences. Such high conservation of the 3'UTRs suggests a specialized and significant function for this region in mammals.

3' Untranslated Regions↗
PubMed1967

Reaction of myoglobin with 3,3-tetramethyleneglutaric anhydride.

1. The extent of reaction of the protein with 3,3-tetramethyleneglutaric anhydride was measured by determining the loss of lysine and the equivalent appearance of 3,3-tetramethyleneglutarimidolysine in the hydrolysate at various time-intervals. Of the 19 lysine residues in sperm-whale myoglobin, three did not react with 3,3-tetramethyleneglutaric anhydride. Also, the N-terminal valine did not react. 2. Slight spectral changes were observed on modification of myoglobin. 3. Electrophoretic changes were great since the derivative was negative throughout the range pH6-8.8. 4. The derivative did not react with antisera to native sperm-whale myoglobin.

Amino Acids↗
PubMed1996

Aplysia limacina myoglobin cDNA cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis.

The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back transplanting the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)-containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved.

Amino Acid Sequence↗
PubMed1998

Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R.

As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two-colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single-cell suspensions of thymus, lymph node and spleen. Anticetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220 x 10(3) MW, with the 180 x 10(3) MW from being predominantly expressed on T cells and the 220 x 10(3) MW form expressed predominantly on B cells and thymocytes F21.H labelled all B cells and a proportion of T cells on single-cell suspensions of spleen cells. CD45R- killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.

Animals↗