The primary structure of the hemoglobin of spectacled bear (Tremarctos ornatus, Carnivora)

The complete primary structure of the alpha- and beta-chains of the hemoglobin of Spectacled Bear (Tremarctos ornatus) is presented. Following cleavage of the heme-protein link and chain separation by RP-HPLC, their amino-acid sequences were determined by Edman degradation in liquid- and gas-phase sequenators. The hemoglobin of Spectacled Bear displays only five amino-acid exchanges to that of Polar Bear (Ursus maritimus, Ursinae) and Asiatic Black Bear (Ursus tibetanus, Ursinae) whereas 8 and 12 replacements, respectively, to Giant Panda (Ailuropoda melanoleuca) and Lesser Panda (Ailurus fulgens) can be found. This clearly demonstrates that the Spectacled Bear, the most aberrant bear of the Ursidae, is somewhat intermediate between Pandas and Ursinae.     

Primary structure of hemoglobin of the polar bear (Ursus maritimus, Carnivora) and the Asiatic black bear (Ursus tibetanus, Carnivora

The adult Polar and Asiatic Black Bear have one hemoglobin component each. The complete amino-acid sequences of their alpha- and beta-chains are presented. Their primary structures were determined by sequencing the tryptic and prolyl peptides. The alignment of these peptides was deduced from homology to human hemoglobin chains. The hemoglobin sequences of the two species proved to be identical. The evolutionary aspects of this result are discussed. A table of identical hemoglobin sequences from different species is given.     

粪便取样对亚洲黑熊脑源性神经营养因子基因全长序列分析

沿用本室改进的粪便提取方法,参照马来熊BDNF基因序列设计引物,首次从亚洲黑熊粪便DNA中扩增和克隆到包含完整核BDNF基因的753 bp片段,以毛发作阳性对照并进行重复实验,获得稳定一致结果。序列分析表明,亚洲黑熊的BDNF基因非常保守,与人相比,一致性达94.5%,与大熊猫比达98.9%。在推导的多肽序列中,其成熟区氨基酸序列与所有已报道哺乳动物的完全一致;对亚洲黑熊及其相关物种BDNF基因序列的比较分析,发现大熊猫与包括黑熊在内的熊科动物亲缘关系更近,而与小熊猫较远。文章首次采用非损伤性取样法在分子生物学水平对亚洲黑熊基因组核BDNF基因进行分析,不仅为亚洲黑熊的保护和繁育提供重要参考资料,为非损伤性取样在珍稀濒危野生动物研究中的应用拓宽了思路,也为亚洲黑熊及其近缘种的系统分类研究提供分子证据。     

长白山自然保护区黑熊和棕熊种群数量动态分析

1986～2010年冬季采用路线调查方法,对吉林省长白山国家级自然保护区阔叶红松林和针叶林中的黑熊(Ursus thibetanus)及棕熊(U.arctos)的相对种群数量和幼年个体比例进行了长期调查。调查面积分别为4万和3万hm2。调查期间累计遇到黑熊65头次(2.6头/年)、棕熊46头次(1.8头/年)。各年度黑熊和棕熊的数量变动很大,总体呈下降趋势。黑熊的数量较20世纪80年代下降了93.4%,幼体所占比率从17.2%下降为0%;棕熊种群数量下降了38.8%,幼体所占比率仅为7.1%。黑熊和棕熊的种群幼体所占比例极低,说明长白山自然保护区黑熊和棕熊自然繁殖力很低,2种熊种群均处于极度濒危状态。栖息地减少和盗猎可能是导致熊类数量急剧下降的主要因素。     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.     

Predictive Modeling and Mapping of Malayan Sun Bear (Helarctos malayanus) Distribution Using Maximum Entropy

One of the available tools for mapping the geographical distribution and potential suitable habitats is species distribution models. These techniques are very helpful for finding poorly known distributions of species in poorly sampled areas, such as the tropics. Maximum Entropy (MaxEnt) is a recently developed modeling method that can be successfully calibrated using a relatively small number of records. In this research, the MaxEnt model was applied to describe the distribution and identify the key factors shaping the potential distribution of the vulnerable Malayan Sun Bear (Helarctos malayanus) in one of the main remaining habitats in Peninsular Malaysia. MaxEnt results showed that even though Malaysian sun bear habitat is tied with tropical evergreen forests, it lives in a marginal threshold of bio-climatic variables. On the other hand, current protected area networks within Peninsular Malaysia do not cover most of the sun bears potential suitable habitats. Assuming that the predicted suitability map covers sun bears actual distribution, future climate change, forest degradation and illegal hunting could potentially severely affect the sun bear’s population.     

Longitudinal monitoring of fecal testosterone in male Malayan Sun bears (U. malayanus)

Fecal steroid monitoring was applied as a non‐invasive method to investigate testicular cycles and seasonality in the Malayan Sun bear (Ursus malayanus), an endangered ursid from South East Asia. Fecal testosterone was analyzed by radioimmunoassay in samples collected from male Sun bears (n=8) housed in zoological parks in North America and New Zealand, over periods of <27 months. Testosterone levels were often, but not exclusively, elevated during mating periods with peaks accompanying breeding behavior and copulation. There was a significant effect of age with older bears having clearly higher concentrations of fecal testosterone (P<0.001). Testosterone concentrations fluctuated throughout the year, with no significant effect of season (P>0.05). All bears did, however, share a common pattern of annual excretion that suggests a potential role for non‐photoperiodic seasonal influences on testicular cycles. Levels were generally lower early in the year with regular increases occurring at 3–4‐month intervals. Grouped data suggest an association between cycles of testosterone production in males and months of peak reproductive activity in captivity. Zoo Biol 0:1–15, 2005. © 2005 Wiley‐Liss, Inc.     

Oxygen binding and other physical properties of human hemoglobin made in yeast.

Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.Abbreviations used GMHX the holo-protein (including b-type heme, Glycera dibranchiata monomer hemoglobin Component X (X=2, 3, or 4) - GMGX the apo-protein, or globin, Glycera dibranchiata monomer globin derived from Component X (X=2, 3, or 4) - rec-gmg the globin derived from a recombinant holoprotein of a Glycera dibranchiata monomer hemoglobin, rec-gmh, whose sequence has been inferred from an isolated cDNA insert - CB label refers to peptides generated from cyanogen bromide cleavage of GMG4 - HPLC high-performance liquid chromatography - T label refers to peptides generated from trypsin digests of GMG4 - Mb myoglobin - MCS monomer hemoglobin crystal structure from Glycera dibranchiata. H, N-terminal sequence of GMG4 - SWMb sperm whale myoglobin     

Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.     

Specific antibodies to hemoglobin A1 (anti-Glu) and hemoglobin S (anti-Val) in the guinea pig: immunologic and structural correlations.

Like goats and sheep, guinea pigs can produce, in response to human sickle cell hemoglobin (beta6 Glu leads to Val), an antibody population (anti-Val) that will bind sickle cell hemoglobin but not normal hemoglobin HbA. Unlike goats and sheep, guinea pigs can produce in response to human hemoglobin A1 an antibody fraction, anti-Glu, that will not react with human sickle cell hemoglobin. These anti-Glu antibodies have been isolated by affinity chromatography and their specificity confirmed by fluorescence-quenching titrations. The sequence of the first 10 amino acids of the beta-chain of guinea pig hemoglobin has been determined. This sequence differs from those of both hemoglobin HbA and sickle cell hemoglobin by two residues, those at positions 5 and 6. This explains the similarity of the immunogenicity of this site on the two human hemoglobins when administered to guinea pigs. Both goats and sheep are identical to hemoglobin A1 at the beta-6 position.     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Comparative Analysis and Molecular Characterization of a Gene BANF1 Encoded a DNA-Binding Protein During Mitosis from the Giant Panda and Black Bear

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin.

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tubulopapillary mammary carcinoma in a brown bear (Ursus arctos)

A 28-yr-old, nulliparous female brown bear (Ursus arctos) at the Karacabey Ovakurusu Bear Sanctuary presented with an enlargement of the mammary gland. Three other nodules were also noted in the proximity of the mammary gland and over the vulva. Clinical, hematologic, ultrasonographic, and radiologic examinations were performed; the enlarged mammary gland was removed and the other masses were also excised. Histopathologic examination revealed tubulopapillary carcinoma of the mammary gland, and the other masses were diagnosed as epidermoid cysts. This is the first reported case of tubulopapillary mammary carcinoma accompanied by epidermoid cysts in a bear.     

A precursor to hemoglobin mRNA in nuclei of immature duck red blood cells

A method is described for sedimenting RNA on a preparative scale in the presence of 85% formamide. It is likely that the RNA is fully denatured, since under the same conditions ribosomal RNA develops full hyperchromicity, and formaldehyde-treated and untreated ribosomal and nuclear RNA sediment in an identical manner.RNA from immature duck red blood cells was fractionated on sucrose gradients. The amount of hemoglobin mRNA and poly(A) in each fraction was measured by hybridization with complementary DNA and poly(U), respectively. We observed that hemoglobin mRNA forms aggregates in the presence of phenol, which are dispersed on formamide gradients. Nuclear RNA became more heavily aggregated, and again the aggregates were dispersed by formamide. A possible nuclear precursor of hemoglobin mRNA was identified. The molecular weight of the precursor is 6–7 × 10⁵, three times as long as hemoglobin mRNA, and it is attached covalently to a poly(A) sequence.     

Biosynthesis and amino terminal acetylation of cat hemoglobin B

Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β^B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β^B globin also occurs in the completed hemoglobin.     

Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate.

Two opioid peptides were isolated from a bovine hemoglobin hydrolysate, by use of gel permeation (GP) and reverse phase (RP) high performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. The same fragments occur in human hemoglobin in positions 32-41 and 33-41 of the beta-chain, respectively. The opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea-pig ileum (GPI), were significant and comparable with some others previously described. In addition, the location of the two opioid peptides, VV-hemorphin-7 and LVV-hemorphin-7, revealed the existence of a "strategic zone" both in the bovine and human beta-chains of hemoglobin.