Duck Erythrocytes Research Articles

Functional separation of the Na-K exchange pump from the volume controlling mechanism in enlarged duck red cells.

Previous publications have described a "volume controlling mechanism" in duck erythrocytes that returns both enlarged and shrunken cells to their original isotonic volume. Enlarged cells return to their original size by readjusting their K content. To study the specificity of this aspect of the mechanism for K, we prepared enlarged cells with various Na and K contents. Only cells containing a high K content resume their original size in the standard isotonic medium. The process of regulation resembles that described above. In contrast, cells containing a high Na content fail to reestablish this volume, but shrink instead until they reach a limiting minimal volume (four-fifths of normal). Here, another mechanism, the cation pump rather than the volume controlling mechanism, removes Na and is responsible for the changes in cell size. Enlarged cells with an intermediate Na and K content utilize both mechanisms to reduce their cation content. Only if Na is prevented from leaving the cell and sufficient K is present initially, will these cells reestablish their original size. These studies demonstrate that the cation pump and volume controlling mechanism function independently and, when cells enlarge, only K can effectively traverse the pathway associated with the volume controlling mechanism. This route differs from the one used by the cation pump to eject Na.

Protochordate immunity: I. Primary immune response of the tunicate Ciona intestinalis to vertebrate erythrocytes

Serum from the tunicate Ciona intestinalis contains a low titer, natural agglutinin for a variety of vertebrate erythrocytes. Primary injections of 8.5 × 10 3 human erythrocytes or 7.0 × 10 2 duck erythrocytes into the tunic tissues or perivisceral cavity were agglutinated and then cleared by phagocytosis within 24 hr. Higher concentrations of human or duck erythrocytes injected into the tunic tissues were encapsulated. Concomitant with clearance or encapsulation, serum agglutinin levels decreased and then returned to normal within 24 hr. Tunicates are classified taxonomically with the vertebrates in the phylum Chordata. Since phagocytosis and encapsulation reactions are typical of most invertebrates, the results of this study suggest that tunicate internal defense mechanisms are more closely allied to invertebrates than to vertebrates.

Incorporation of 14C-amino acids by malarial plasmodia ( Plasmodium lophurae) : VI. Changes in the kinetic constants of amino acid transport during infection

The majority (10 of 17) of amino acids tested entered the mature duck erythrocyte by a saturable, non-uphill transport system, whereas for the erythrocyte-free malarial parasite, Plasmodium lophurae, the converse was true: most amino acids entered the parasite by simple diffusion. Only five amino acids (glutamic and aspartic acids, cysteine, lysine, arginine) showed mediated entry into P. lophurae. The pattern of mediated amino acid transport into the duck erythrocyte was altered upon infection, e.g., either entry was by diffusion or there was a reduced affinity for the amino acid. Transport characteristics similar to those found in the malaria-infected erythrocyte were produced by treating normal duck red cells with a cell-free extract of malaria-infected erythrocytes and quinine (a depressor of red cell ATP). It is suggested that depletion of host cell ATP as well as elaboration of as yet unidentified substances by the parasite promote the changes in permeability seen in the malaria-infected cell.

Cell-free translation of the globin message within polydisperse high-molecular-weight ribonucleic acid of avian erythrocytes.

Polydisperse high-molecular-weight RNA of nucleated avian erythrocytes includes sequences coding for globin chains. The RNA was extracted from immature erythrocytes of ducks and fractionated under denaturing conditions by sucrose density gradient centrifugation in 99% dimethylsulfoxide. The RNA sedimenting faster than 45 S was able to direct the synthesis of duck globins in the Krebs II ascites cell-free protein-synthesizing system. The newly synthesized globin molecules have been identified by their characteristic electrophoretic properties in polyacrylamide gels containing either urea or sodium dodecyl sulfate, and by immunoprecipitation of the released globin chains by rabbit antibodies against duck hemoglobin. In order to rule out the possibility of a contamination of the high-molecular-weight RNA with duck-globin messenger RNA tailing from the 9-10S region, rabbit-globin messenger RNA was added to duck RNA as an internal control. No rabbit-globin messenger RNA activity could be detected in the RNA fractions sedimenting faster than 45 S. It is concluded that high-molecular-weight RNAs in the nucleated erythroid cell contain sequences of globin messenger RNAs covalently attached to larger polynucleotide chains. These results support the view that polydisperse nuclear RNA is the precursor of the cytoplasmic messenger RNA fraction.

Fine structure of the malaria parasite Plasmodium lophurae developing extracellularly in vitro.

SYNOPSISDuck malaria parasites (Plasmodium lophurae), synchronized at the uninucleate trophozoite stage, were freed from their host erythrocytes by immune lysis and cultured extracellularly in duck erythrocyte extract medium. At 0 time, 1, 2, and 3 days, samples were taken for light and electron microscopy and for measurement of incorporation of [14C]‐methionine or [14C]‐proline. For 2 days the parasites developed fairly normally, progressing from large trophozoites‐early schizonts at 1 day to segmenters‐forming merozoites at 2 days. However, the 3‐day samples showed signs of deterioration: incorporation of amino acids dropped; the percentage degenerate cells rose; the progression of developmental stages slowed. At the fine structure level 2 abnormalities were observed which may indicate the limits of extracellular cultivation in vitro. Through 2 days of culture all parasites were surrounded by 2 membranes. The 3‐day samples contained some organisms with only one membrane, which may have arisen from merozoites produced extracellularly. The 2nd alteration was in the food vacuoles, which were progressively fewer, smaller, and less dense in the cultured samples and may indicate an abnormality in the extracellular parasite's feeding mechanism.

The response of duck erythrocytes to norepinephrine and an elevated extracellular potassium. Volume regulation in isotonic media.

This paper presents evidence that duck erythrocytes regulate their size in isotonic media by utilizing a previously reported "volume-controlling mechanism." Two different experimental situations are examined. In the first, cells enlarge in a solution containing norepinephrine and an elevated [K](o); and in the second, enlarged cells shrink to their original size if the norepinephrine and excess potassium are removed. As the erythrocytes enlarge, K, Cl, and H(2)O accumulate. Shrinkage, in contrast, is accompanied by the controlled loss of K, Cl, and H(2)O. These changes and the associated changes in membrane permeability resemble those reported previously when duck erythrocytes incubate in anisotonic media. There cells, after first shrinking or swelling, utilize a "volume-controlling mechanism" to reestablish their original size. The mechanism regulates cell size by adjusting the total number of osmotically active intracellular particles. The present studies indicate duck red cells use this mechanism to readjust their total monovalent cation content and thus their solute content in isotonic media as well. In addition, evidence is presented which indicates that the "volume-controlling mechanism" and ouabain-inhibitable cation pump differ functionally.

Identification of free cytoplasmic globin mRNA of duck erythroblasts by hybridization to anti-messenger DNA and by cell-free protein synthesis

The cytoplasm of animal cells contains mRNA not associated with ribosomes which exists in the form of ribonucleoprotein (RNP) complexes [ 1,2] . It has been suggested that such complexes represent a pool of mRNA intermediate between the nuclear pre-mRNA and the mRNP complex active in translation [l-4] . We have described in immature duck erythrocytes a cytoplasmic 9 S RNA not associated with ribosomes. This free cytoplasmic 9 S RNA has the same electrophoretic mobility as polyribosoma19 S globin mRNA; its rate of synthesis and decay is consistent with, but not a proof of, a role as a precursor to polyribosomal 9 S mRNA [4] . In this paper we present direct proof for the existence of a cytoplasmic pool of genuine globin mRNA free from the ribosomes (free mRNA) by demonstrating the capacity of the free 9 S RNA to be translated into globins and to hybridize with anti-messenger DNA (amDNA) copied from globin mRNA by reverse transcriptase.

Polyribosome-bound and free-cytoplasmic-hemoglobin-messenger RNA in differentiating avian erythroblasts.

Cytoplasmic messenger‐like RNA not associated with ribosomes has been investigated in immature duck erythrocytes and compared with polyribosome‐bound messenger RNA. It is shown that 9‐S RNA of the same electrophoretic mobility as the polyribosomal 9‐S globin messenger is present free in the cytoplasm. Its rate of synthesis and decay is consistent with a role as a precursor of polyribosomal 9‐S globin messenger.mRNA species different from 9‐S RNA, among them a predominant 12‐S species, are found associated with polyribosomes as well as in the free cytoplasmic pool. The free cytoplasmic mlRNA pool is more heterogeneous compared to the polyribosomal mRNAs, suggesting a translational control qualitatively and quantitatively selecting among the free cytoplasmic mlRNAs for those to be translated.

The non-histone proteins of chromatin: Their isolation and composition in a number of tissues

1. 1. A method is described for the fractionation of salt-urea-dissociated chromatin using hydroxylapatite. With the exception of experiments using chromatins prepared from “citric acid” nuclei, high yields of acidic non-histone proteins, relatively free of RNA, can be obtained by this procedure. 2. 2. The non-histone proteins of a number of chromatins were compared by electrophoresis in sodium dodecyl sulphate-urea polyacrylamide gels employing a discontinuous buffer system. Proteins from mouse chromatins prepared from “citric acid” nuclei were found to be extremely heterogeneous, but in the case of calf thymus the proteins were mainly low molecular weight. On the other hand, the non-histone proteins of chromatin from “sucrose” nuclei appeared to contain fewer high molecular weight species in the tissues studied, with the exception of brain. Preparation of nuclei by the double-detergent procedure of Penman ( J. Mol. Biol., 17 (1966) 117) gave chromatin with a low protein to DNA ratio. These proteins also appeared to be predominantly low molecular weight. Duck erythrocyte nuclei prepared by lysis also contained low molecular weight chromatin non-histone proteins. 3. 3. Using salt fractionation techniques attempts were also made to remove “cytoplasmic” and “residual” acidic proteins from chromatin. The proteins which remained with the DNA and histones were found to be mainly low molecular weight in kidney and liver, but in the case of brain a wide spectrum of proteins was seen. 4. 4. Little tissue or species specificity of non-histone proteins were found on comparison of “sucrose” nuclei chromatins prepared from a number of mouse and bovine tissues. 5. 5. It is concluded that the non-histone proteins which remain tightly bound to DNA in chromatin are of the same approximate size as the basic histones. Because of the procedural variation in the heterogeneity of these chromatin proteins, detection of single species of regulatory proteins appears to require other techniques.

Purine uptake and utilization by the avian malaria parasite Plasmodium lophurae.

SYNOPSIS. Plasmodium lophurae cannot carry out extensive de novo purine biosynthesis, and depends upon the host erythrocyte for a supply of preformed purines. Exogenous purines taken up by the parasitized erythrocyte may constitute a major source of preformed purines for parasite nucleotide biosynthesis. The uptake of exogenous radioactive purine compounds and their incorporation into nucleic acids by duck erythrocytes parasitized with P. lophurae, uninfected erythrocytes, and erythrocyte‐free parasites were studied. P. lophurae was found to have a remarkable ability, both intracellularly and extracellularly, to take up and utilize certain exogenous purines such as adenosine, inosine, and hypoxanthine. Incorporation studies indicated that this species has a functional purine salvage pathway by which inosine, hypoxanthine, and adenosine can be converted to both adenine and guanine nucleotides.

Two Chemically and Metabolically Distinct Forms of Calf Thymus Histone F3

Abstract The F3 histones of duck erythrocytes and calf thymus have been purified and compared by a variety of techniques. The results of analytical gel electrophoresis, gel filtration, cysteine content determinations, and amino acid analysis support the conclusion that there exist two chemically and metabolically distinct forms of F3 histone. The duck erythrocyte has a single form of F3 histone, which contains 1 cysteine residue. The calf thymus, however, has two forms of F3 histone. The minor form present as approximately 20% of the total F3 histone fraction closely resembles the duck erythrocyte F3 in that it contains a single cysteine residue. The major form of calf thymus F3 histone, however, contains 2 half-cystine residues. These two forms of F3 histone differ metabolically in that they are acetylated at different rates in isolated calf thymus nuclei. The implications of these findings are discussed in terms of the possible evolutionary origins of histone F3.

Differential turnover of two messengers in one cell type: 9S globin mRNA and 12S mRNA in differentiating avian erythroblasts

The kinetics of synthesis and decay of 9S globin mRNA and 12S mRNA isolated from the same polyribosomes was investigated in immature duck erythrocytes. It is shown that the two messengers have a different stability in vivo. The rate of synthesis of 12S mRNA is high in cell populations rich in early erythroblasts. In populations containing more cells in the later stages of erythrocyte maturation the 9S globin mRNA is more rapidly synthesized than 12S mRNA. During an actinomycin D chase the 12S mRNA decays faster than the 9S globin mRNA. The rate of synthesis and decay of 12S and 9S mRNA as well as their different base composition implies the existence of two independent messenger species rather than a precursor-product relationship.

Changes in the neutral glycosyl ceramides of duck erythrocytes during infection with the avian malaria parasite, [formula omitted][formula omitted

Utilization of thin layer chromatography for comparison of erythrocyte glycosphingolipids resulted in the detection of qualitative similarities but quantitative dissimilarities between malarious and non-malarious ducklings. Levels of type 1 (GC1) and a hematoside-like (GCH) neutral glycosyl ceramide decreased markedly in animals infected with Plasmodium lophurae ; little variation was noted in GC3b; slight increases were observed in GC2 and GC3a. Possible differences may relate to the presence of a parasite neuraminidase.

Specifically Cytotoxic Lymphocytes Produced by Preincubation with Antibody-Complexed Target Cells

Summary Highly purified human blood lymphocytes which had been incubated with antibody-complexed chicken erythrocytes, adsorbed 50 to 100 × more of this antibody (rabbit) than lymphocytes incubated with antibody-IgG without erythrocytes. Most of the adsorbed IgG was present in the form of antigen-antibody complexes, derived from lysed erythrocytes and not removable from the lymphocytes by repeated washing. When tested in subsequent cytotoxicity tests, the lymphocytes which carried antigen-antibody complexes were specifically cytotoxic for freshly added chicken erythrocytes. This was not the case with lymphocytes preincubated with antibody-IgG alone, with antibody IgG and noncross-reactive duck erythrocytes, or with IgG from normal rabbit serum. The results indicate that some lymphocytes have high affinity for antigen-antibody complexes. Antibody taken up in this way may be reutilized, thereby conferring specific effector functions on a population of normal lymphocytes. When complex-carrying lymphocytes were incubated with fresh erythrocytes and small amounts of fresh anti-erythrocyte antibody, however, they were less cytotoxic than those preincubated with antibody alone or with normal rabbit IgG. This may at least in part reflect a competition for lymphocytic sites with affinity for immunoglobulin and may be necessary for triggering of the antibody-induced cytotoxicity of lymphocytes.

The response of duck erythrocytes to hypertonic media. Further evidence for a volume-controlling mechanism.

The addition of a hypertonic bathing medium to duck erythrocytes results in an initial instantaneous phase of osmotic shrinkage and, when the [K](o) of the hypertonic solution is larger than "normal," in a second, more prolonged phase, the volume regulatory phase. During the latter, which also requires extracellular Na, the cells swell until they approach their initial isotonic volume. The increase in cell volume during the volume regulatory phase is accomplished by a gain in the cell content of K, Cl, and H(2)O. There is also a smaller increase in the Na content of the cell. Potassium is accumulated against an electrochemical gradient and is therefore actively transported into the cell. This accumulation is associated with an increase, although dissimilar, in both K influx and efflux. Changes in cell size during the volume regulatory phase are not altered by 10(-4)M ouabain, although this concentration of ouabain does change the cellular cation content. The response is independent of any effect of norepinephrine. The changes in cell size during the volume regulatory phase are discussed as the product of a volume controlling mechanism identical in principle to the one reported in the previous paper which controls cell volume in hypotonic media. Similarly, this mechanism can regulate cell size, when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.

The response of duck erythrocytes to nonhemolytic hypotonic media. Evidence for a volume-controlling mechanism.

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10(-4)M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.

Alterations in sodium and potassium in red blood cells and plasma during the malaria infection ( Plasmodium lophurae)

1. 1. During the course of infection of Plasmodium lophurae (avian malaria) in white Pekin ducklings the red blood cell sodium concentration was significantly elevated in parasitized animals (14·0±1·32 mM; mean ± S.E.M.) as compared to normal animals (8·8 ± 0·72 mM); the plasma potassium level was also significantly increased in malarious ducklings (5·4 ± 0·29 mM) when compared to normal ducklings (2·9 ± 0·18 mM). 2. 2. Cation concentrations of P. lophurae more closely resembled those of the duck erythrocyte than of duck plasma.

Malaria parasites (Plasmodium lophurae) developing extracellularly in vitro: incorporation of labeled precursors.

SYNOPSIS. P. lophurae were removed from their host duck erythrocytes and incubated in vitro in certain modifications of the red cell extract medium previously described. The extent of incorporation, into material precipitable with trichloracetic acid, of 14C‐labeled precursors supplied after 15–16 hr of incubation, was determined and compared with effects on structure of the parasites. A decreased concentration of erythrocyte extract, which always resulted in increased numbers of degenerate parasites and decreased development to multinucleate forms, also decreased the incorporation of methionine‐methyl‐14C and orotic‐acid‐6‐14C. It did not affect incorporation of proline‐U‐14C or of choline‐1,2‐14C. With a 1/3rd strength red cell extract, omission of coenzyme A, which increased the proportion of degenerate parasites and usually decreased the multinucleate forms, decreased the incorporation of all 4 substrates, in keeping with the inability of the parasites to synthesize CoA. On the other hand, omission of ATP and pyruvate, which had an even greater deleterious effect on structure of the parasites than omission of CoA, had no effect on incorporation of methionine or orotic acid and probably none on that of choline. Incorporation of adenine was reduced in presence of ATP or AMP, suggesting competition at an uptake site. Incorporation of proline, however, was higher with ATP and pyruvate present, in keeping with the better development of the extracellular parasites. The uptake of proline may depend on an ATPase in the outer of the 2 membranes surrounding the parasite.

The Effect of Norepinephrine and Dibutyryl Cyclic Adenosine Monophosphate on Cation Transport in Duck Erythrocytes

Freshly prepared duck erythrocytes, incubated either in plasma or an isotonic synthetic medium containing norepinephrine ([K] of both media approximately 2.5 mM), maintain water and electrolyte composition in the steady state (upper steady state) for at least 90 min. If incubated in the synthetic medium without norepinephrine or in plasma to which a beta-adrenergic blocking agent (propranolol) is added, the cells lose both water and electrolyte (predominantly KCl) until a new steady state is reached (lower steady state). Reaccumulation of water and electrolyte from isotonic solutions toward the upper steady-state levels requires the addition of norepinephrine and KCl. Reaccumulation is maximal when the concentration of K and norepinephrine in the medium is 15 mM and 10(-7)M, respectively. Dibutyryl cyclic-AMP (10(-2)M) mimics norepinephrine in lower steady-state cells. Although an analogous effect in upper steady-state cells was not established with certainty, it is proposed that the catecholamine-induced net changes in water and electrolyte movement in duck erythrocytes are a consequence of stimulation of the activity of a membrane-bound adenyl cyclase system.

Effects of X-Irradiation on Hemoglobin Polymorphism in the Adult Pekin Duck

The rates of synthesis and concentrations in blood of the two hemoglobins found in adult duck erythrocytes were measured periodically during several months after x-irradiation of the animal. Labeled glycine incorporation into the heme and globin components of the hemoglobins and labeled plasma iron incorporation into the heme components in blood in vitro were used to estimate the rates. The rates for both hemoglobins decreased during days 0-2 postirradiation and remained depressed until day 11. The depression in rate between days 7-11 was greater for the major than for the minor hemoglobin. Between days 11-25, the rates increased. The increase was less for the major than for the minor hemoglobin. After day 25, the rates returned to about the initial values. The changes in the rates were accompanied by roughly parallel changes in the blood reticulocyte level. The concentrations of the hemoglobins in blood decreased during days 5-15 and increased between days 15-30. The increase in concentration was less fo...