Nucleated Chicken Erythrocytes Research Articles

Toxoplasma gondii adhesion and apoptosis of chicken erythrocytes

Toxoplasma gondii is thought to infect all nucleated cells in warm-blooded animals, including poultry, mammals, and humans. However, it is unclear whether T. gondii can infect chicken erythrocytes due to the nucleated nature of these cells. Due to the special role of chicken erythrocytes in innate immunity, we investigated the cell‒cell interaction between T. gondii and erythrocytes to elucidate the role of chicken erythrocytes in T. gondii infection. Cellular apoptosis was analyzed by transwell assay and flow cytometry. An immunofluorescence method was used to examine the reorganization of vimentin during T. gondii infection in both Vero cells and chicken erythrocytes. The reorganization of actin was evaluated to further examine the invasion capacity of tachyzoites on chicken erythrocytes during infection. We discovered that T. gondii can adhere to but not invade chicken erythrocytes and eventually cause apoptosis in chicken erythrocytes. When tachyzoites were cocultured with chicken erythrocytes in vitro, the transcriptional levels of T. gondii MIC3, ROP16, and ROP18 were significantly decreased. In addition, the rearrangement of host cell vimentin, a type III cytoskeleton protein regulated by T. gondii infection, was not observed. Similarly, the parasite-induced ring-shaped actin structure was not formed in the host-parasite junction. T. gondii (RH strain) tachyzoites preferentially invaded Vero cells and replicated in chicken blood monocytes, but they were not found in chicken erythrocytes. These findings showed that although T. gondii could attach to the surface of chicken erythrocytes, but couldn’t invade successfully. Interestingly, we found that the T. gondii secretome, lysates, and intact tachyzoites could cause apoptosis of chicken erythrocytes, which suggested a complex mechanism involved in the apoptosis of chicken erythrocytes induced by T. gondii. This study elucidated that T. gondii could not infect nucleated chicken erythrocytes and enriched our understanding of the transmission mechanism of T. gondii among avian species.

Further characterization of cation channels present in the chicken red blood cell membrane

In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K + saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd 3+ and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.

The Effects of Ionizing Radiation on Avian Erythrocytes

The effects of ionizing radiation were examined in terminally differentiated cells using nucleated chicken erythrocytes (RBCs) as the model. We used a hemolytic assay to score radiation damage to RBCs. Chicken RBCs received 0 to 100 Gy of radiation at dose rate of 10 Gy/min. Radiation-induced hemolysis occurred in a dose-dependent manner but not immediately after irradiation. Hemolysis became apparent at 24 h after treatment. A threshold for radiation dose response was observed. At doses below 30 Gy, hemolysis in irradiated samples was indistinguishable from that in nonirradiated controls. A total dose of 100 Gy was used for the split-dose experiments. The results showed that chicken RBCs were able to repair radiation damage and that the half-time for maximum recovery was approximately 30 min at 36 degrees C. Recovery from gamma radiation was also affected by the interfraction temperature.

A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis

The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chromatin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and repressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone H5 as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termination stage-specific protein) is hyperexpressed at the terminal stage of chicken erythropoiesis and is accumulated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It appeared to be a basic polypeptide (p I 9.2) which, however, precipitated at low pH. When reconstituted in vitro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleo-somes, thus mimicking the processes occuring in vivo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correlation with their transcriptional activity. No other basic proteins (H5, cytochrome c, RNase A) added to the nuclear preparation at the same level as MENT (protein/ DNA = 0.005) caused any effect on nuclear organization. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei having little or no histone H5. We propose that MENT cooperates with histone H5 to complete the nuclear collapse in mature nucleated erythrocytes.

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.

Role of plasma membrane and of cytomatrix in maintenance of intracellular to extracellular ion gradients in chicken erythrocytes.

Ultrastructural observations in combination with electron probe X-ray microanalysis on detergent (Brij 58) permeabilized (disruption of the plasma membrane) nucleated chicken erythrocytes support the view that a large fraction of cytoplasmic and nuclear K+ is not freely diffusible and that adsorption of K+ on detergent released mobilizable proteins exists within the cell. The data also suggest that the detergent proteins are normally immobilized by a detergent-resistant cytoskeleton so that they are not immediately free to diffuse from the cell for several minutes after detergent disruption of the plasma membrane.

Identification of both calpains I and II in nucleated chicken erythrocytes.

Chicken erythrocytes were found to contain two species of calpains which differ in elution profile from DEAE-cellulose and in Ca2+ requirement. After partial purification, one of them was half-maximally activated by 10 microM Ca2+ and the other by 180 microM Ca2+. The low- and high-Ca2+-requiring proteases cross-reacted only with the respective monospecific antibodies for mammalian calpain I and calpain II, respectively. Approximately 5 times more calpain I than calpain II is present in chicken erythrocytes. By immunoelectrophoretic blot analysis, both calpains I and II from chicken erythrocytes were proved to be heterodimers composed of 76 and 28 kDa, and 80 and 28 kDa subunits, respectively. Our present finding that the heavy subunit of calpain I is smaller than that of calpain II is noteworthy, since the opposite is known to be true of various mammalian calpains. An immunological study has revealed that the calpain I newly found in chicken erythrocytes is not derived from calpain II. Thus, the co-existence of calpains I and II in one animal species also holds in chickens, contrary to the previously advocated notion that chickens have only one type of calpain.

Effects of immunization of mothers on the immune reaction of their offspring: inhibition of immune responses of offspring caused by antibody imported through the milk.

Effects of immunization of pregnant AKR mice with nucleated chicken erythrocytes (CRBC) on immune responses of their offspring were examined. Antigen-specific reduction of generation of cytotoxicity and plaque forming cells (PFC) was demonstrated in the offspring at 8 weeks after birth, and lasted for 15 weeks. Cross-fostering experiments and cell transfer experiments showed that such suppression would be induced by antibody contained in the milk of immunized mothers rather than suppressor cells. Activities to enhance opsonization and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) were demonstrated in the serum of such offspring before challenge with CRBC. Delayed footpad reaction (DFR) was maintained at the normal level in such offspring of immunized mice.

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo.

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.

Restoration by levamisole of immune responses suppressed in tumor-bearing mice.

Restorative effects of levamisole on suppressed immune responses were observed in BALB/c mice inoculated with a syngeneic methylcholanthrene-induced fibrosarcoma and immunized with nucleated chicken erythrocytes. In tumor-bearing mice, cell-mediated cytotoxicity, delayed footpad reaction, and antibodies were suppressed, and only the delayed footpad reaction was restored by levamisole. Suppressive effects in tumor-bearing mice can be transferred with sera or spleen cells, and the expression of suppressive effects cannot be abolished by treatment of the recipients with levamisole. However, the generation of suppressor mechanisms was affected by treatment of tumor-bearing donors with levamisole.

Deoxycytidine excretion by mouse peritoneal macrophages: its implication in modulation of immunological functions.

Pyrimidine excretion by macrophages was studied in order to identify the potential immunoregulatory effector molecules. Deoxycytidine was found in the culture medium of thioglycollate-elicited mouse peritoneal macrophages, along with thymidine, which was shown by others to be a possible immunoregulatory substance. The identification of deoxycytidine was based on: (1) cochromatography with the authentic compound in four different solvents, (2) UV absorption spectral analysis, and (3) the enzymatic peak shift method. Phagocytosis of nucleated chicken erythrocytes, but not enucleated sheep erythrocytes, increased deoxycytidine excretion. The macrophages lacked both deoxycytidine kinase and deoxycytidine deaminase, which is consistent with their excretory pattern. Since it has been known that deoxycytidine can protect cells against cytotoxic effects of thymidine, we propose that deoxycytidine has a role in preventing immunosuppression by thimidine. In patients with adenosine deaminase deficiency, however, immunosuppression caused by combined toxicity of thymidine and deoxyadenosine may not be adequately prevented by deoxycytidine.

Nucleus-anchoring cytoskeleton in chicken red blood cells

Cytoskeleton of chicken erythrocytes was studied after extraction of the cells with Triton X-100. In phase contrast microscopy the extracted cells were seen as ghost-like structures with preserved morphology, distinct nucleus and surrounding plasma membrane remnant. In electron microscopy, dense matrix-like nucleus, fibrillar plasma membrane residue and filaments, ca. 10 nm in diameter, traversing the cytoplasmic domain, were seen. Distinct bands of molecular weight 68000, 53000, 32000 and some bands of both higher and lower molecular weight were obtained by polyacrylamide gel electrophoresis of the extracted cells. These results indicate that intermediate filaments, forming the nucleus-anchoring cytoskeleton, are present in nucleated chicken erythrocytes as part of cellular cytoskeleton.

Enzyme loading of nucleated chicken erythrocytes

Exogenous enzymes can be loaded into nucleated chicken erythrocytes by hypotonic hemolysis. The hemolysed and resealed cells retain the ability to activate transport ions and to survive while circulating in the blood of chickens. It is proposed that these cells may be useful for the investigation of several aspects of cellular biochemistry.

Lymphoblastic lymphoma of convoluted or acid phosphatase type-a tumor of T precursor cells.

Five lymphatic neoplasms with strong focal acid phosphatase reactivity were selected from a group of acute lymphocytic leukemias and lymphoblastic lymphomas. All five cases showed an anterior mediastinal mass and exhibited identical morphology. This type of lymphoma has been described by Lukes under the term "malignant lymphoma of convoluted lymphocytes". Analysis of surface membrane receptors revealed that the tumor cells lacked surface immunoglobulin and receptors for Fc-fragment, but possessed receptors for complement (C3), untreated SRBC (ES) and SRBC treated with neuraminidase (ESN). By applying a mixed rosette assay using nucleated chicken erythrocytes coated with antibodies and C3, and denucleated ESN, it was found that a considerable number of tumor cells in all five cases formed mixed rosettes, i.e. that they bore the C3 receptor characteristic of B cells and simultaneously the E receptor characteristic of T cells. Thus the tumor cells resembled immature thymocytes of 10-15 weeks' gestation, which also show focal acid phosphatase reactivity and simultaneous expression of C3 and E receptors.

HEMOLYSIS OF CHICKEN BLOOD

1. The time-dilution curves are given for the hemolytic action of saponin, sodium taurocholate, and sodium oleate on nucleated chicken erythrocytes. 2. Saponin and sodium taurocholate cause hemolysis but leave the nuclei and ghosts in suspension, thereby making the end-point of hemolysis more arbitrary than the clear end-point for non-nucleated cell hemolysis. 3. The curves of hemolysis by saponin and taurocholate are shown to be of the same nature as are found in the hemolysis of non-nucleated cells. 4. Sodium oleate causes first hemolysis and then, in the stronger solutions, causes karyolysis. Two pairs of values for kappa and c = infinity are thus obtainable for the same reaction, one pair for the destruction of corpuscular membrane, the other pair for the destruction of the nucleus. 5. Viscosity changes are found in the lysin-cell system with strong concentrations of sodium taurocholate and sodium oleate. Time-viscosity curves are given for these changes. 6. Microscopically, the action of these lysins on the nucleated chicken red cell appears to be similar to their action on the non-nucleated erythrocytes.