Hepatic or splenic targeting of carrier erythrocytes: a murine model

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H2O2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved.     

Irreversible AE1 tyrosine phosphorylation leads to membrane vesiculation in G6PD deficient red cells

Background

While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs.

Methodology/Principal Findings

Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation.

Conclusions/Significance

Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products.     

A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells

Background

Imaging single cells with fluorescent markers over multiple cell cycles is a powerful tool for unraveling the mechanism and dynamics of the cell cycle. Over the past ten years, microfluidic techniques in cell biology have emerged that allow for good control of growth environment. Yet the control and quantification of transient gene expression in unperturbed dividing cells has received less attention.

Methodology/Principal Findings

Here, we describe a microfluidic flow cell to grow Saccharomyces Cerevisiae for more than 8 generations (≈12 hrs) starting with single cells, with controlled flow of the growth medium. This setup provides two important features: first, cells are tightly confined and grow in a remarkably planar array. The pedigree can thus be determined and single-cell fluorescence measured with 3 minutes resolution for all cells, as a founder cell grows to a micro-colony of more than 200 cells. Second, we can trigger and calibrate rapid and transient gene expression using reversible administration of inducers that control the GAL1 or MET3 promoters. We then show that periodic 10–20 minutes gene induction pulses can drive many cell division cycles with complete coherence across the cell cluster, with either a G1/S trigger (cln1 cln2 cln3 MET3-CLN2) or a mitotic trigger (cdc20 GALL-CDC20).

Conclusions/Significance

In addition to evident cell cycle applications, this device can be used to directly measure the amount and duration of any fluorescently scorable signal-transduction or gene-induction response over a long time period. The system allows direct correlation of cell history (e.g., hysteresis or epigenetics) or cell cycle position with the measured response.     

Effects of uncoupling of mitochondrial energy conservation on the ultradian clock-driven oscillations in Saccharomyces cerevisiae continuous culture

Protonophores have several different perturbative effects on dissolved O2 concentrations in continuous cultures of Saccharomyces cerevisiae. As well as uncoupling energy conservation from mitochondrial electron transport in vivo, they reset ultradian clock-driven respiratory oscillations and produce cell cycle effects. Thus, additions at low concentration (1.25 microM) of either m-chlorocarbonyl-cyanide phenylhydrazone (CCCP) or 5-chloro-3-t-butyl-2-chloro-4(1)-nitrosalicylanilide (S13) led to phase resetting of the 48 min ultradian clock-driven respiratory oscillations. At 2.5 microM CCCP or 4 microM S13, transient inhibition of oscillatory respiration (for 5 h) preceded synchronisation of the cell division cycle seen as a slow (9 h period) wave that enveloped the 48 min oscillation. At still higher concentrations of CCCP (5 microM), the cell division cycle was prolonged by about 7 h, and during this phase, the respiratory oscillation became undetectable. The significance of these observations with respect to the time-keeping functions of the ultradian clock is discussed.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes

Rats were injected with⁵⁹Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2–7.4 in the former case and 6.4–6.7 in the latter. Red cells were then centrifuged (5) and approximately 7–10% of the packed cells from the top and 7–10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2–7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4–6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4–6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Changes in the respiratory properties of the blood in the carp,Cyprinus carpio, induced by diurnal variation in ambient oxygen tension

Summary Effects of diurnal variation in ambient oxygen tension on acid-base balance and blood respiratory properties were investigated in carp (Cyprinus carpio). The carp were subjected to two cycles in ambient between about 130 mm Hg and about 12 mm Hg at 17°C (cf. Figs. 1 and 2). The first period of hypoxia was characterized by a non-compensated respiratory alkalosis, i.e. whole blood showed an increase in pH from 7.92 to 8.14. During the second hypoxic period, 24 h later, a significantly smaller respiratory alkalosis was present, whole blood pH changed from 7.95 (the value found during the intermediate return to normoxia) to 8.08. The latter increase was associated with a significant increase of 25% in plasma bicarbonate concentration compared with the first period of hypoxia (Fig. 1, Table 1). The erythrocytic concentrations of hemoglobin and ATP were lowered by about 10%, compared with the normoxic values, during the two episodes of hypoxia, and this was due to a swelling of the erythrocytes during hypoxia. The red cell GTP concentration showed an altogether different change during the O₂ cycling: the absolute concentration of red cell GTP changed to a steady level, 50% below that present at the onset of the experiment and the major part of this change took place between the two hypoxic periods (Fig. 2, Table 2).The results are discussed with reference to the respiratory function of the blood of carps during subjection to cyclic changes in nature.     

Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes

Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).     

Activation of the Na,K-pump by isoproterenol, methylxanthines, and iodoacetate in erythrocytes of the frogRana temporaria

Our preliminary studies have shown that the Na,K-pump in frog erythrocytes is activated by isoproterenol (ISP), phosphodiesterase blocker (3-isobutyl-methylxantine, IBMX), and by iodoacetate (MIA). The aim of the present study was to determine a mechanism responsible for the effect of MIA on the Na,K-pump activity in frog red blood cells as well as the role of G proteins and intracellular messengers in modulation of active K⁺ transport induced by ISP. An additive stimulation of active K⁺ (⁸⁶Rb) transport in frog erythrocytes was found after exposure of the cells to MIA in a combination with ISP or IBMX. The treatment of the red blood cells with 1 mM MIA for 1 or 2 h was associated with a significant decrease in intracellular Na⁺ concentration, on average, by 13 and 20%, respectively, suggesting a direct action of MIA on the Na,K-pump. Incubation of cells in the presence of dibutyryl-cAMP (1 mM) or adenylate cyclase activator forskolin (0.1 mM) caused stimulation of the active K⁺ influx by 21.8 and 27.9%, respectively. AlF ₄ ^- and cholera toxin able to increase cell cAMP levels via G protein interactions had no effect on the total and IPS-induced K⁺ influx in frog erythrocytes. The treatment of the red blood cells with sodium nitroprusside that increases cGMP concentration in cells also had no effect on the K⁺ influx. The stimulatory influence of ISP on the Na,K-pump was reduced with increase of the intracellular Na⁺ concentration. ISP increased affinity of the Na,K-pump to Na⁺ (the Mihaelis constant K_M = 34.4 ± 5.1 in control and 25.3 ± 2.8 mM in the presence of ISP,p < 0.01), but did not change maximal velocity (8.1 ± 0.6 and 7.7 ± 0.3 mmol/1/h in the control and ISP-treated cells, respectively). The results obtained indicate the presence of several different signal pathways involved in regulation of the Na,K-pump activity in frog erythrocytes.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3sigma and CDKN1A (p21) knockout cell lines

Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3sigma increased the amount of cell death at 96 h, from 52% to approximately 70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3sigma expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and anti-apoptosis function, while 14-3-3sigma has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05-1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3sigma resulted in an increase in apoptosis and a decrease in survival.     

Regulation of self-recognition by T-cell hybrids

Somatic cell hybrids were prepared between BW 5147, an AKR T lymphoma, and purified T cells from three sources: spleen cells exposed to sheep red blood cells, lymph node cells from mice sensitized to ovalbumin, and spleen cells of mice injected with azobenzenearsonate-IgG. Hybrid lines expressed constitutive markers of both parents which include H-2 antigens and the isoenzymes glucose phosphate isomerase and isocitrate dehydrogenase. Furthermore, they expressed both parental alleles of Thy 1, a differentiation antigen. Many of the hybrid lines formed rosettes with mouse erythrocytes. T-cell hybrids did not bind human or chicken red blood cells, though they did rosette with sheep erythrocytes to the same extent as with mouse red cells. We interpret the latter reaction as due to recognition of shared antigens by the murine T cells. This form of self-recognition is influenced by culture conditions and is expressed optimally by cells in late logarithmic phase of growth.     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

The response of trout red cells to adrenaline during seasonal acclimation and changes in temperature

Adult rainbow trout were acclimated to three different temperature and photoperiod regimes: 17°C, 14 h light: 10 h dark (summer); 7° C, 14 h light: 10 h dark; and 5° C, 8 h light: 16 h dark (winter). Blood was collected from these fish after 40 days acclimation, and the response of red blood cells to in vitro adrenergic stimulation was assessed. To examine potential seasonal variations in endogenous levels of circulating catecholamines, plasma levels of adrenaline (Ad) and noradrenaline (NAd) were measured at rest and after exercise. At rest, there were no differences between groups in plasma levels of either Ad or NAd, but, after exercise, the pattern of catecholamine elevation differed. In fish acclimated to 17 and 7° C in summer, Ad and NAd increased by about the same amount (10–15 times). In fish acclimated to 5° C in winter, NAd increased about three-fold, compared to the near 50-fold increase in Ad levels. Whether this difference between groups can be attributed to seasonal influences is unclear. At both low (0·5%) and high (2%) PCO ₂, adrenergic stimulation (2 × 10^-7 M Ad) of trout red cells in vitro led to a significant reduction in MCHC (mean cell [haemoglobin]), compared to non-stimulated cells. However, only at the high PCO ₂ were pHe and red cell pHi significantly different from those in the non-stimulated cells: the latter was higher and the former lower in the stimulated cells. There were no differences in the response of red cells to adrenergic stimulation between groups of fish. Under the conditions of the present study no influence of season and/or temperature on the in vitro response of trout red cells to adrenergic stimulation was shown.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.     

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K⁺ influx measured as ⁸⁶Rb uptake for 60 min. The Na⁺-K⁺-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1–2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K⁺ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K⁺ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K⁺ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01–0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K⁺ influx into frog erythrocytes. These results indicate that about one-half of the Na⁺ -K⁺-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate “membrane-associated” ATP pool. Accepted: 12 July 1997     

Copper activation of superoxide dismutase in rat erythrocytes

Several lines of evidence suggested that copper can activate a preexisting pool of superoxide dismutase (SOD) apoprotein in erythrocytes from copper-deficient rats. First, feeding adequate copper to copper-deficient rats raised initially low erythrocyte SOD activities to normal values in under one-third the time needed to replace the entire red cell population. Moreover, copper injection (1 mg Cu/kg, sc) doubled erythrocyte SOD activity levels in 16 h. Since protein synthesis is restricted in mature erythrocytes, these results imply that copper activated apoSOD in vivo. Furthermore, injected copper raised SOD activity contents of both young and old erythrocytes. Neither dietary copper status nor copper injection influenced red cell SOD immunoreactive protein levels. In contrast, copper injection increased the amount of copper associated with the SOD activity peak region resulting from gel filtration of hemoglobin-free erythrocyte proteins on Sephadex G-75. Copper ions (3 microM) elevated SOD activity levels in vitro by 63% in 4 h in intact red cells from copper-deficient rats. No activation took place in lysed red cells from the same rats or in intact cells from copper-adequate rats. These results all suggest that copper can activate SOD apoprotein in erythrocytes by a specific, saturable process.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Circadian clock regulation of the bimodal rhythm of cyclic AMP in wild-type Euglena

Cell-cycle traverse is associated with fluctuations in the cellular content of cAMP; artificial alterations of these levels phase-shift cell division in free-running cultures of achlorophyllous Euglena maintained in constant darkness (DD). The phase shifts observed, however, are only transient: the cell division rhythm rephases to that of unperturbed controls. This implies that the second messenger functions downstream of the circadian oscillator. Further, the level of cAMP is known to indicate carbon nutrient status and the competency of cells to traverse various restriction points in the cell cycle of other eukaryotes. We wished to determine the profile of cAMP content in free-running, dividing and non-dividing cultures of green, wild-type cells, which survive well during prolonged growth arrest. We monitored cAMP content in photoautothropic cultures of E. gracilis (strain Z) at 25 degrees C under either an entraining light-dark cycle comprising 12 h of light and 12 h of darkness (LD:12,12) or free-running (LD:1/2,1/2) regimes. cAMP content in rhythmically dividing, light-phased or free-running cells exhibited bimodality [peaks at CT (circadian time) 9-14 and CT 19-22). Expression of cAMP content on a per milligram total cellular protein basis caused the day trough (CT 1-3) to be even more distinct. Non-dividing, free-running, photoautotrophic cultures displayed a similarly phased bimodality in cAMP content. These findings in wild-type Euglena confirm that the bimodal rhythm of cAMP content is regulated by the circadian oscillator that underlies division rhythmicity but is not dependent on the cell division cycle. We will now determine the effect of the fluctuating cAMP levels on the phosphorylation status and activity of cell-cycle regulatory proteins.