Duck Erythrocytes Research Articles

Identification of genetic determinants of hemolytic activity of Riemerella anatipestifer using random transposon mutagenesis

Riemerella anatipestifer causes epizootic infectious disease in poultry resulting in serious economic losses especially to the duck industry. In our previous study, R. anatipestifer was found to lyse duck erythrocytes in vitro. In the present study, a random Tn4351 mutagenesis library of hemolytic R. anatipestifer strain SX containing 4000 mutants was constructed to investigate the genetic basis of hemolytic activity. Thirty mutants with reduced hemolytic activity and one with increased hemolytic activity were screened and insertions in 24 genes were identified. Of these genes, four were predicted to encode outer membrane proteins, one encoded a cytoplasmic membrane protein, 11 encoded cytoplasmic proteins, and eight encoded proteins with unknown locations. Based on current annotations of the R. anatipestifer genomes, of the 24 genes, 7 (29.17%) were involved in iron utilization. The hemolytic activities of the complemented strains M2 (pRES-Riean_0790) and M18 (pRES-Riean_0653) were restored, indicating that both Riean_0653 and Riean_0790 are involved in the hemolytic activity of strain SX. However, the recombinant proteins rRiean_0317, rRiean_0790, rRiean_0653, rRiean_1027, rRiean_1143, and rRiean_1561 had no hemolytic activity, suggesting that none were hemolysins.

Characterization of the hemolytic activity of Riemerella anatipestifer.

Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R. anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba+ (with hemolytic activity on duck blood agar) and dba- strains created pores in the duck red blood cells, with 4.35-9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba+R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba+ and dba-R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

Morphometric Parameters of Erythrocytes in Juvenile Mandarin Ducks Aix galericulata

ABSTRACT The morphometric parameters of erythrocytes in Mandarin ducks- Aix galericulata have never been subjected to study even though this species is common in aviculture. The aim of this study is to investigate the morphometric parameters of erythrocytes in juvenile Mandarin ducks. A total of 1500 erythrocytes obtained from thirty Mandarin ducks were analysed. The smears of the peripheral blood were stained using Diff-Quik® stain method, and the length and width of erythrocytes were measured using ImageJ program. The average length of the measured erythrocytes was 11.879 µm. The maximal measured length of the erythrocytes was 16.049 µm, and the minimal measured length was 5.668 µm. The average width of the measured erythrocytes was 6.160 µm. The maximal measured width of the erythrocytes was 10.115 µm and the minimal measured width was 3.750 µm. In conclusion, knowing morphometric parameters of Mandarin duck erythrocytes is the first step in evaluation process of erythrocytes’ functional state and health condition of an individual.

The progressive effects of a high‐fat diet on erythrocyte osmotic fragility, growth performance and serum triglyceride and cholesterol levels in Guinea fowl (Numida meleagris) and Muscovy duck (Cairina moschata)

To investigate the progressive effects of a high-fat diet on erythrocyte osmotic fragility, growth performance and serum lipid concentrations in Guinea fowl and Muscovy ducks, 36 Guinea fowl and 36 Muscovy ducks were divided into two groups, for each species, and fed either a standard (STD=commercial poultry feed) or high-fat diet (HFD=commercial poultry feed with 20% palm oil and 2% lard) for up to 12weeks. After 4, 8 and 12weeks on the diets, six birds from each group were euthanized and blood samples collected. Osmotic fragility was assessed by measuring the haemoglobin released by erythrocytes placed in serially diluted solutions of phosphate-buffered saline, spectrophotometrically. Serum triglyceride and cholesterol concentrations were also determined. Fragiligrams from erythrocytes from both species of birds on the HFD were not different to those on the STD. However, Muscovy duck erythrocytes were more resistant to haemolysis compared with Guinea fowl erythrocytes. Final body mass and serum triglyceride levels were not significantly different (p>0.05, anova) between the birds in the HFD and STD groups, for both species of birds. In contrast, serum cholesterol levels were significantly higher in birds on the HFD compared with those on the STD, after 4, 8 and 12weeks of feeding, for both species of birds. Feeding Guinea fowl and Muscovy ducks a high-fat diet for up to 12weeks resulted in hypercholesterolaemia but had no effect on final body mass, erythrocyte osmotic fragility or serum triglyceride concentrations in either bird species.

Linker histone‐like proteins in Muscovy duck (Cairina moschata L) erythrocyte chromatin

Linker Histone-Like proteins (LHL1 and LHL2) were identified within a linker histone complement of Muscovy duck erythrocyte chromatin. Polyacrylamide gel electrophoretic patterns of N-bromosuccinimide-cleaved LHL products as well as liquid chromatography-electrospray-ion trap mass spectrometry analyses of trypsin-digested LHL peptides revealed structural similarity of LHL1 to histone H5 and between LHL2 and histone H1 subtypes. Since the LHL proteins were stable in the presence of 2-mercaptoethanol and dithiothreitol that reduce disulfide bonds, it appeared unlikely that this doublet was a thiol-derived product of linker histones. A loss of LHL1, with a concomitant maintenance of LHL2 after treatment with dilute alkali, seems to suggest that they might represent disparate protein conjugates resulting from linker histone modifications through ester linkages.

Nucleic acid distribution pattern in avian erythrocytes and mammalian lymphocytes: Comparative studies by fluorescence microscopy and digital imaging analytical techniques

Nucleated erythrocytes of healthy domestic chicken and ducks, and lymphocytes of healthy Sprague Dawley rats were evaluated for nucleic acid distribution pattern, employing light and fluorescence microscopy procedures, as well as digital imaging analytical methods. The results demonstrate a unique organization of nuclear DNA of mature chicken and duck erythrocytes, as well as immature duck erythrocytes, as delineated spherical nuclear bodies that mostly corresponded with euchromatin zones of the cells in routine Wright-stain blood smears. The nuclear DNA of the rat lymphocytes, on the other hand, was observed as a more diffuse green fluorescing nuclear areas, with punctate variably-sized diffuse areas of RNA red fluorescence. RNA red color fluorescence was also evident in the narrow cytoplasm of the lymphocytes, especially in large lymphocytes, in comparison with the cytoplasm of the mature avian erythrocytes that completely lacked any nucleic acid fluorescence. Nuclear RNA fluorescence was lacking in the mature chicken erythrocytes, compared with those of the mature and immature duck erythrocytes as well as lymphocytes of both avian and rats blood. The significance of these findings lies in the establishment of normal benchmarks for the nuclear and cytoplasmic nucleic acid pattern in eukaryotic cells. These normal benchmarks become valuable in rapid diagnostic situations associated with pathologies, such as the presence of viral nuclear and cytoplasmic inclusion bodies that can alter the nucleic acid pattern of the host cells, and in conditions of cellular abnormal protein aggregations. Variability of cellular nucleic acid pattern can also aid in prognostic assessments of neoplastic conditions.

Genetic polymorphism of histone H1.b in duck erythrocytes

Six different phenotypes of erythrocyte histone H1.b in ducks were described by using two-dimensional polyacrylamide gel electrophoresis. The inheritance data were consistent with the occurrence of three codominant alleles of H1.b at an autosomal locus. Each of the alleles gave rise to one fraction. Two of the alleles showed a frequency of approximately 0.5 each in all of the three lines studied. The third allele was present in only one line with a frequency of 0.08. The analysis of samples by only one-dimensional acid-area polyacrylamide gel electrophoresis resulted in insufficient separation and did not enable the scoring or H1.b variants.

Impact of Zn, Cu, and Fe on the Activity of Carbonic Anhydrase of Erythrocytes in Ducks

The impact of zinc, copper, and iron on the duck erythrocyte carbonic anhydrase (CA) activity and the hemoglobin content in vitro culture were studied. The increase of zinc or iron addition at a low level induced the rise of CA activity, and the CA activity was inhibited by zinc or iron at a high addition level. The duck erythrocyte CA was strongly inhibited by cupric ion. The inhibition constant of duck erythrocyte CA to cupric ion is about 3.5 microM. Carbonic anhydrase compared to hemoglobin is more sensitive to zinc and copper in the environment. These findings suggest that some characteristics of duck erythrocyte CA are different from both CAI and CAII of mammals. The increase of Fe addition below 8 microM in the minimal essential medium brought about the rise of CA activity and resulted in the maximum of CA activity exceeding that induced by Zn. It provided a new evidence for the role of ferrous ion in CA.

In vitro susceptibility of duck, chicken, and pig erythrocyte lipids to peroxidation

The susceptibility of erythrocyte lipid to in vitro peroxidation as measured by TBARS (thiobarbituric acid-reactive substances) and concentration of reduced glutathione (GSH) in whole blood and erythrocyte suspension before and after incubation with hydrogen peroxide was assessed in ducks, chickens and pigs. A high susceptibility of erythrocytes to peroxidation in vitro was observed in all animals tested, but this susceptibility was of different intensity. Pig erythrocytes exhibited the lowest resistance to oxidative stress in vitro as compared with that in ducks (P < 0.01) and chickens (P < 0.02). A high level of GSH in the erythrocytes of ducks and chickens offers higher resistance to oxidative stress in comparison with that in the pig erythrocytes.

Natural allelic variation of duck erythrocyte histone H1b

In our previous work (J. Pałyga, Genetic polymorphisms of histone H1.b in duck erythrocytes. Hereditas 114, 85–89, 1991) we reported a genetic polymorphism of duck erythrocyte histone H1.b. Here, we screened H1 preparations in a two-dimensional polyacrylamide gel to refine the distribution of allelic forms of H1.b in fifteen duck populations. We have revealed that the frequency of H1.b allelic variants was significantly different among many conservative and breeding duck groups. While b 1 and b 3 were common in all populations screened, the allele b 2, with a slightly lower apparent molecular weight, was confined mainly to brown-feathered ducks (Khaki Campbell and Orpington) and descendent lines. The C- and N-terminal peptides released upon cleavage with N-bromosuccinimide and Staphylococcus aureus protease V8 from duck allelic histones H1.b2 and H1.b3, respectively, migrated differently in the gel, probably as a result of potential amino acid variation in a C-terminal domain.

Allelic polymorphism of histone H1.a in duck erythrocytes.

Allelic polymorphism of histone H1.a in duck erythrocytes.

A quantitative description of the Na-K-2Cl cotransporter and its conformity to experimental data.

In epithelia, the Na-K-2Cl cotransporter cooperates with other transport mechanisms to produce transepithelial NaCl transport. The reaction cycle for the Na-K-2Cl cotransporter has been established experimentally, but whether it accounts, quantitatively, for experimental findings has yet to be established. The differential equations that describe the reaction cycle were formulated, and the steady-state solutions were obtained by digital computation. Conformity between this description and the experimental data obtained from the literature was explored by automatic searches for the sets of rate constants that yielded statistical best-fits to the experimental data. Fits were obtained from renal epithelial cell lines, HeLa cells, and duck erythrocytes. Results show that the reaction cycle for the Na-K-2Cl cotransporter conforms well, quantitatively, with the experimental data.

Activation of the Avian Erythrocyte Na-K-Cl Cotransport Protein by Cell Shrinkage, cAMP, Fluoride, and Calyculin-A Involves Phosphorylation at Common Sites

Na-K-Cl cotransport activity in duck erythrocytes increases approximately 10-fold in response to osmotic cell shrinkage, norepinephrine, fluoride, or calyculin-A (an inhibitor of type-1 and -2a phosphatases). To assess whether all four stimuli promote phosphorylation of the cotransport protein and whether this phosphorylation is catalyzed by the same kinase, the cotransporter was isolated from erythrocytes by immunoprecipitation and its pattern of phosphorylation was evaluated. Each stimulus evoked proportionate increases in cotransporter activity and phosphorylation. No two stimuli in combination evoked greater activation and phosphorylation than did the more potent of the two stimuli acting alone. Phosphoamino acid analysis of the cotransport protein indicated that phosphorylation occurs at serine and threonine residues. Phosphopeptide mapping revealed a distinctive pattern of 8 major tryptic phosphopeptides, none of which were significantly phosphorylated in the unstimulated state. Maps of cotransporters activated by the four different stimuli were indistinguishable. Measurements of phosphorylation stoichiometry indicated that each cotransporter acquires approximately 5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. Staurosporine, a kinase inhibitor with broad selectivity, inhibited each stimulus equipotently (IC50 approximately 0.7 microM). Staurosporine promptly reversed cotransporter activity and phosphorylation when added to shrinkage-stimulated but not to calyculin-stimulated cells, indicating that it enters the cell rapidly and blocks phosphorylation. These results suggest that cell shrinkage, cAMP, fluoride, and calyculin-A promote the phosphorylation of the Na-K-Cl cotransport protein at a similar constellation of serine and threonine residues. It is proposed that all modes of stimulation ultimately involve the same protein kinase.

Effect of transmembrane Ca2+ gradient on the coupling of beta-adrenergic receptors and adenylyl cyclase.

In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of beta-AR and adenylyl cyclase, beta-AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2+ gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated beta-AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of beta-AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled beta-AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted beta-AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated beta-AR via Gs.

Protein-induced bending or flexing at the 5′-end of the duck β A-globin promoter

The 5′-end of the duck β A-globin promoter contains a protein binding site BS-3/Spl, the A + T-rich part of which could be involved in DNA bending. Plasmids were constructed using the pBend2 plasmid containing BS-3/Spl. Circular permutation analysis of the fragments cut out from the plasmids using various restriction endonucleases, in the presence of a partially purified protein extract from embryonic duck erythrocytes, was performed. The results indicate that a rather complicated change in the fragment shape takes place upon protein binding, which is best explained as an induction of two points of bending or flexure within the fragment. Analogical points of flexure may exist at the protein-binding sites of the duck and chicken β A-globin promoters in spite of differing DNA sequences.

Genetic polymorphism of histone H1.z in duck erythrocytes

Three different phenotypes of erythrocyte histone H1.z were detected in several lines of duck by using two-dimensional PAGE. Electrophoresis and inheritance data have shown that two co-dominant alleles, z1 and z2, encode two proteins which differ slightly in their apparent molecular masses. Allele z1 was very abundant and was found at a frequency at least 0.94, while allele z2 was very rare and was present at a frequency of less than 0.06. Limited chemical and enzymic cleavages appeared to indicate that the allelic forms of histone H1.z differed in the C-terminal regions of their molecules.

Transcription factor IIA is inactivated during terminal differentiation of avian erythroid cells.

Avian histone H5 and alpha A-globin genes are transcribed much more efficiently in whole cell extracts derived from immature polychromatic erythrocytes than in extracts from mature duck erythrocytes. We found that these differential activities are detectable only if assayed with promoters containing a functional TATA box. The addition of either highly purified human or recombinant yeast transcription factor IIA (TFIIA) to extracts from mature erythrocytes resulted in a significant increase in transcription from TATA-containing promoters, whereas transcription from TATA-less promoters remained unaffected. Moreover, the activity of TFIIA was found to be reduced in extracts from mature erythrocytes. These data support the proposition that inactivation of TFIIA may contribute to a general repression of gene activity in avian erythrocytes, and only those genes with alternative mechanisms of initiation complex formation continue to be expressed in these cells. In the case of the histone H5 gene, such an alternative mechanism could be mediated via the interaction between duck erythrocyte upstream stimulating factor and TFIID.

Transcription factor eUSF is an essential component of isolated transcription complexes on the duck histone H5 gene and it mediates the interaction of TFIID with a TATA-deficient promoter

We analysed the formation of transcription complexes on the H5 gene of the duck which is efficiently transcribed in HeLa cell extracts in vitro. Upon deletion of its TATA-box, the fidelity of transcription of the H5 gene is maintained, although the efficiency of this process is significantly reduced. Selective inactivation of TFIID in whole cell extracts and reconstitution experiments either with human recombinant TFIID or a protein fraction from duck erythrocytes enriched in TFIID show that transcription of the TATA-less H5 promoter nevertheless requires the protein TFIID. Screening of promoter elements which could indirectly mediate the interaction of TFIID with a TATA-less H5 promoter led to the identification of a sequence element located about 40 base-pairs downstream from the H5 initiation site that shows partial homology to the USF consensus sequence. In electrophoretic mobility shift and footprinting studies we demonstrated a specific interaction of the erythroid factor USF (eUSF) with this downstream element. By isolating active transcription complexes we found that all components required for correct initiation remain stably associated with the H5 promoter irrespective of the presence or absence of the TATA box. Moreover, the reconstitution of eUSF and TFIID-depleted transcription complexes with purified protein fractions demonstrate that not only TFIID but also eUSF essentially participates in complex formation even on H5 promoter mutations lacking the TATA-box. Mutual interactions between eUSF and TFIID appear to stabilize the binding of TFIID in the presence or absence of its proper binding site.

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: Selectivity and kinetic properties of ion binding sites

The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport-activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [3H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na+ and K+ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The Km for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na+ and K+ binding sites. Na+ and K+ were partially replaceable, with the selectivity of the Na+ site being Na+ greater than Li+ greater than NH4+; N-methyl-D-glucamine+, choline+ and tetramethylammonium+ also supported a small amount of specific binding when substituted for Na+. The selectivity of the K+ site was K+ approximately Rb+ greater than NH4+ greater than Cs+; N-methyl-D-glucamine+, choline+ and tetramethylammonium+ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li+ could partially substitute for Na+ in supporting cotransport, but other monovalent cations were completely inactive. The order of potency at the K+ site was NH4+ greater than K+ approximately Rb+ greater than Cs+ much greater than other monovalent cations. The effect of Cl- on bumetanide binding was biphasic, being stimulatory at low [Cl-] but inhibitory at high [Cl-]. As this implies the existence of two Cl- binding sites (termed ClH and ClL for the "high-" and "low-" affinity sites, respectively) each phase was examined individually. Cl- binding to ClH could be described by a rectangular hyperbola with a Km of 2.5 mM, while kinetic analysis of the inhibition of bumetanide binding at high [Cl-] revealed that it was of a noncompetitive type (Ki = 112.9 mM). The selectivity of anion binding to the two sites was distinct. ClH was highly selective with Cl- greater than SCN- greater than Br-; F-, NO3-, ClO4-, MeSO4-, gluconate- and SO4(2-) were inactive. The efficacy of anion inhibition of binding to ClL was ClO4- greater than I- greater than SCN- greater than NO3- greater than Cl-; F-, MeSO4-, gluconate-, and SO4(2-) were inactive. Thus, ClH is much more selective than ClL and largely accounts for the specificity of the system with respect to anion transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein-binding A + T-rich motifs flank the duck β A-globin enhancer

The duck β A-globin (β GLB) enhancer DNA was analysed by footprinting for sites of specific binding of proteins extracted from duck erythrocytes. The results were compared with previously determined protein binding to the homologous region in chicken DNA. Two A + T-rich protein-binding sites, not recognized in chicken, were found at the 5'-end and the 3'-end of the duck β GLB enhancer. The 5'-motif (designated BS-1; 5'-AAACAAAATGAA) binds proteins extracted from both embryonic and adult erythrocytes, while the 3 '-motif (55-2 ; 5'-ATAAACAAGGTC) binds protein from embryonic cells only.