Multi-qubit entanglement and algorithms on a neutral-atom quantum computer.

Gate-model quantum computers promise to solve currently intractable computational problems if they can be operated at scale with long coherence times and high-fidelity logic. Neutral-atom hyperfine qubits provide inherent scalability owing to their identical characteristics, long coherence times and ability to be trapped in dense, multidimensional arrays1. Combined with the strong entangling interactions provided by Rydberg states2-4, all the necessary characteristics for quantum computation are available. Here we demonstrate several quantum algorithms on a programmable gate-model neutral-atom quantum computer in an architecture based on individual addressing of single atoms with tightly focused optical beams scanned across a two-dimensional array of qubits. Preparation of entangled Greenberger-Horne-Zeilinger (GHZ) states5 with up to six qubits, quantum phase estimation for a chemistry problem6 and the quantum approximate optimization algorithm (QAOA)7 for the maximum cut (MaxCut) graph problem are demonstrated. These results highlight the emergent capability of neutral-atom qubit arrays for universal, programmable quantum computation, as well as preparation of non-classical states of use for quantum-enhanced sensing.

High purity single photons entangled with an atomic qubit.

Trapped atomic ions are an ideal candidate for quantum network nodes, with long-lived identical qubit memories that can be locally entangled through their Coulomb interaction and remotely entangled through photonic channels. The integrity of this photonic interface is generally reliant on the purity of single photons produced by the quantum memory. Here, we demonstrate a single-photon source for quantum networking based on a trapped 138Ba+ ion with a single photon purity of g (2)(0)=(8.1±2.3)×10-5 without background subtraction. We further optimize the tradeoff between the photonic generation rate and the memory-photon entanglement fidelity for the case of polarization photonic qubits by tailoring the spatial mode of the collected light.

Rydberg-Mediated Entanglement in a Two-Dimensional Neutral Atom Qubit Array.

We demonstrate high fidelity two-qubit Rydberg blockade and entanglement on a pair of sites in a large two-dimensional qubit array. The qubit array is defined by a grid of blue detuned lines of light with 121 sites for trapping atomic qubits. Improved experimental methods have increased the observed Bell state fidelity to F_{Bell}=0.86(2). Accounting for errors in state preparation and measurement we infer a fidelity of F_{Bell}^{-SPAM}=0.88. Accounting for errors in single qubit operations we infer that a Bell state created with the Rydberg mediated C_{Z} gate has a fidelity of F_{Bell}^{C_{Z}}=0.89. Comparison with a detailed error model based on quantum process matrices indicates that finite atom temperature and laser noise are the dominant error sources contributing to the observed gate infidelity.

Multispecies Trapped-Ion Node for Quantum Networking.

Trapped atomic ions are a leading platform for quantum information networks, with long-lived identical qubit memories that can be locally entangled through their Coulomb interaction and remotely entangled through photonic channels. However, performing both local and remote operations in a single node of a quantum network requires extreme isolation between spectator qubit memories and qubits associated with the photonic interface. We achieve this isolation by cotrapping ^{171}Yb^{+} and ^{138}Ba^{+} qubits. We further demonstrate the ingredients of a scalable ion trap network node with two distinct experiments that consist of entangling the mixed species qubit pair through their collective motion and entangling a ^{138}Ba^{+} qubit with an emitted visible photon.

Randomized benchmarking of single-qubit gates in a 2D array of neutral-atom qubits.

We characterize single-qubit Clifford gate operations with randomized benchmarking in a 2D array of neutral-atom qubits and demonstrate global and site selected gates with high fidelity. An average fidelity of F2=0.9983(14) is measured for global microwave-driven gates applied to a 49-qubit array. Single-site gates are implemented with a focused laser beam to Stark shift the microwaves into resonance at a selected site. At Stark selected single sites we observe F2=0.9923(7) and an average spin-flip crosstalk error at other sites of 0.002(9).

Chemotactic peptide-induced changes in neutrophil actin conformation.

The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.

Rheology of leukocytes, leukocyte suspensions, and blood in leukemia. Possible relationship to clinical manifestations.

Suspensions of leukemic lymphocytes and myeloblasts and blood of leukemic patients were studied to examine (a) the effect of leukemic cells on blood viscosity and (b) the ability of leukemic cells to traverse channels of capillary diameter. The viscosity of suspensions of leukemic cells was dependent logarithmically on (a) shear strain rate and (b) cytocrit, although, suspensions of small lymphocytes and of myeloblasts had a similar viscosity at equivalent shear rates and cytocrit. The minimum apparent viscosity (MAV) of leukemic cells and red blood cells, measured over shear rates of 2.3-230 s(-1) was dependent logarithmically on cytocrit. However, MAV was slightly greater for leukemic cells than for red cells at cytocrits up to 20%. At cytocrits above 20%. MAV of leukemic cells increased more rapidly than that of erythrocytes. For example, at a 15% cytocrit MAV(WBC) (1.85 centipoise) was only slightly greater than MAV(RBC) (1.59); whereas, at 45% cytocrit MAV(WBC) (14.9) was markedly greater than MAV(RBC) (3.81). The blood of subjects with leukemia with marked elevation of leukocyte concentration (leukocrits of 6-32%) had 24% higher mean MAV (3.72) than blood with a similar total cytocrit composed of red cells (3.00). A negative correlation was present between leukocrit and erythrocrit in chronic lymphocytic (r = - 0.82) and chronic granulocytic (r = - 0.81) leukemia. Therefore, the modest increase in whole blood MAV in leukemia can be explained by (a) the negative association of leukocrit and erythrocrit and (b) the rarity of leukocrits over 20% and total cytocrits over 45%. However, the MAV of blood of leukemic patients was 71% greater than expected on the basis of their packed red cell volume. Hence, the ratio of hemoglobin concentration (O(2) carrying capacity) to MAV was abnormally low in the subjects with leukemia studied. Individual leukemic leukocytes were nearly rigid. The mean deformability index (DI) of leukemic myeloblasts (1.22; 1.18) and lymphocytes (1.22; 1.40) as measured by filtration and elastometry, respectively, at 50 mm H(2)O negative pressure, approached that of a rigid body (1.0) as compared to red cells studied by filtration (3.09) or elastometry (4.23). The ability of leukemic cells to traverse nucleopore filter or micropipette channels was related to cell diameter. The relevance of the rheology of leukemic cells to the interruption of blood flow and of tissue oxygen delivery and thereby to clinical manifestations of leukemia is considered.

Potasssium transport in human blood lymphocytes treated with phytohemagglutinin.

We have confirmed that phytohemagglutinin (PHA) rapidly enhances the uptake of potassium (K+) by human blood lymphocytes. PHA, however, did not produce an increase in lymphocyte K+ concentration. The apparent steady-state of cell K+ concentration despite the marked increase in uptake of 42K+ could be explained by either an increase in K+-K+ exchange or an increase in concentrative (active) K+ accumulation in association with an increase in the leak of K+ from the cell. We compared, therefore, the uptake of 42K+ with the decrement in cellular K+ content when active transport was inhibited by ouabain. These studies established that K+-K+ exchange was negligible in human blood lymphocytes and that the increase in 42K+ uptake after PHA treatment represented concentrative transport. Our studies did indicate that 42K+ exodus from PHA treated lymphocytes increased markedly from 19 to 38 mmol-1 cell water-1-h-1. Within the same time period K+ influx into PHA-treated lymphocytes increased from 20 to 38 mmol-1 cell water-1-h-1. Thus, PHA produces a marked increase in the permeability of the lymphocyte membrane to K+, and the increase in active K+ influx in PHA-treated lymphocytes may represent a homeostatic response by the membrane K+ transport system to the increase in K+ efflux. Increased K+ turnover was observed at the lowest concentrations of PHA which produced an observable increase in [3H]thymidine incorporation into DNA. Thus, PHA produces an increase in K+ permeability that closely parallels its mitogenic effect. The rapid increase in K+ influx preceding blastogenesis and mitogenesis is required, therefore, to maintain normal intracellular K+ concentration. An adequate intracellular K+ concentration is essential for the synthetic processes required for cell transformation or division.

Marrow cell egress. The central interaction of barrier pore size and cell maturation.

Release of marrow cells may be determined primarily by the restrictive barrier that separates marrow hematopoietic cords from sinusoids, and by the ability of the cell to negotiate the barrier pores which are of smaller diameter than the cell. This critical interrelationship may be further modulated by humoral agents (releasing factors). To test this hypothesis, we placed human marrow cells in a chamber between millipore or nucleopore filters with pore diameters of 1-8 mum. Fixed, stained, cross sections of the filters allowed histological examination of the penetration of cells and quantification of egress of age-specific cell types. The rate of marrow granulocyte egress was highly correlated with (a) barrier pore diameter. (b) morphological age of cells, and (c) the presence of chemical attractant. Immature granulocytes would not exit through a restrictive barrier even after protracted periods and were not responsive to chemoattractants. Intermediate-aged granulocytes showed a slight ability to respond to attractants and to exit if pore diameters were large. Mature granulocytes exited through the restrictive barrier at all pore diameters studied, however, this egress was accelerated by increasing pore diameter and by the presence of an attractant. Leukemia blast cells were incapable of traversing pore diameters of 1-8 mum.These studies support the hypothesis that the development of deformability, motility, and surface receptors for chemoattractants at the later stages of granulocyte development allow the egress of cells through the marrow sinusoid wall which appears by electron microscopy to be a porous barrier with aperture diameters smaller than cell diameters; and that this process can be modulated by humoral agents which enhance directed movement of cells and may also increase pore size. Moreover, on the basis of our observations, the egress of leukemia cells is best explained by destruction of the normal sinusoid barrier of marrow indicating that manifestations of the disease are dependent on alteration in stromal as well as parenchymal marrow cells.

Calcium exchange and ionized cytoplasmic calcium in resting and activated human monocytes.

We have performed a comprehensive study of calcium tracer flux, distribution, content, and ionized cytoplasmic calcium during monocyte activation. A model of monocyte calcium was developed from 45Ca uptake and exodus curves which indicated that cell calcium was partitioned between three compartments. The magnitude of the time constants for each pool lead us to propose cellular locations for these three compartments: a surface plasma membrane pool, a cytoplasmic pool, and an organelle pool. 45Ca uptake and exodus experiments were analyzed using a nonlinear least squares fit of compartmental exchange rates and sizes. The production of superoxide was used as a reflection of the state of activation of the monocytes treated with Concanavalin A (Con A). We found that Con A-treated monocytes have an increase in the calcium exchange rate with the cytoplasmic pool from 0.04 to 0.07/min (P less than 0.05), and an increase in the size of the cytoplasmic pool from 0.08 to 0.13 pmol/cell (P less than 0.05). There were no significant changes in the exchange rates or sizes associated with either of the other two compartments. The cytoplasmic ionized calcium was measured with the fluorescent probe, Quin 2, which indicated a resting level of 83 nM free calcium in unadhered monocytes. Con A stimulation caused a doubling of the cytoplasmic free calcium to 163 nM within 45 s. This increment in cytoplasmic free calcium preceded the onset of superoxide following Con A treatment. These studies indicate that Con A binding to the plasma membrane increases the monocyte plasma membrane permeability to calcium. External calcium enters the cell at an increased rate and contributes to both internally bound and free calcium. The magnitude of the increase in free calcium is proportional to the concentration of Con A and stimulates calcium extrusion via the calcium transport ATPase. Moreover, there is an increased concentration of ionized cytoplasmic calcium which has the potential to interact with other cellular regulators that modulate cell activation and superoxide production.

Multicomponent analysis of amino acid transport in human lymphocytes. Diminished L-system transport in chronic leukemic B lymphocytes.

We have examined the amino acid transport in B cell chronic lymphocytic leukemia and compared it with the amino acid transport in isolated B lymphocytes from human blood and tonsils. L-system transport was measured with 2-amino-2-carboxy-bicyclo (2,2,1)-heptane, which is a synthetic amino acid whose transport is limited to the L-system. Amino acid uptake was subjected to a multicomponent analysis that partitioned the total uptake into the saturable carrier-mediated transport system and the uptake by diffusion. The maximal velocity of L-system transport in chronic lymphocytic leukemia cells, 81 mumol/1 cell water per min, was less than 10% that of blood B lymphocytes, which was 1,029 mumol/1 cell water per min. The uptake of 2-amino-2-carboxy-bicyclo (2,2,1)-heptane by tonsillar B cells, by a B lymphocyte cell line, and by blood T-lymphocytes was also 10-fold greater than that observed in chronic lymphocytic leukemic cells. Similarly, the L-system uptake of leucine and phenylalanine, which are naturally occurring amino acids usually transported primarily by the L-system, was reduced in chronic lymphocytic leukemic B cells to 15 and 10% of normal B cells, respectively. Total leucine uptake by chronic lymphocytic leukemic cells, however, was sustained at 30% of that expected because of transport via an alternative transport system. The A- or ASC-systems, the other major amino acid transport pathways, were not defective in chronic lymphocytic leukemic cells. These data indicate that there is a specific, profound decrease in L-system carrier-mediated amino acid transport in chronic lymphocytic leukemic B cells, as judged by the system-specific synthetic amino acid, 2-amino-2-carboxy-bicyclo (2,2,1)-heptane. This defect was confirmed by studies with two naturally occurring L-system amino acids, leucine and phenylalanine. This specific abnormality of membrane transport by chronic lymphocytic leukemic B lymphocytes is not shared by other B lymphocyte types, and thus appears to be related to the neoplastic nature of the leukemic B cells rather than to their immunologic subtype.

Relationship of superoxide production to cytoplasmic free calcium in human monocytes.

Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation.

Regulation of sodium and potassium transport in phytohemagglutinin-stimulated human blood lymphocytes.

Phytohemagglutinin (PHA) or concanavalin A treatment of lymphocytes causes an increase in membrane permeability so that the leak rates of Na and K increase 1.5- to 2-fold. Active Na and K transport increase proportionately in response to the increased membrane permeability. We have examined the role of lymphocyte Na concentration in sustaining the increased Na and K transport observed after PHA treatment. Cell Na concentration increases from 14.8 to 20.5 mmol/liter cell water in PHA-treated lymphocytes (P < 0.001). Four lines of evidence suggest that the 5-6 mmol/liter cell water increase in lymphocyte Na accounts for the increase in active Na and K transport in mitogen-treated lymphocytes. First, PHA does not increase directly the maximal Na, K-ATPase activity of isolated lymphocyte membrane vesicles. Second, when the Na concentration is increased by 6 mmol/liter cell water in unstimulated lymphocytes, Na and K transport increase nearly twofold. Third, the cell Na concentration (15 mmol/liter cell water) is near the K(m) for Na activation of the Na, K-ATPase in lymphocyte membranes. The ATPase activity thus, is capable of increasing as the cell Na rises above normal. Fourth, if lymphocytes are incubated in a medium containing a low Na concentration, K transport does not maintain the internal K concentration and the fall in cell K is accentuated in PHA-treated lymphocytes. These studies indicate that the adaptive acceleration of Na and K transport in mitogen-treated lymphocytes is mediated by a small increase in cell Na.

Phorbol ester restores L-system amino acid transport of B lymphocytes in chronic lymphocytic leukemia.

L (leucine-favoring)-system amino acid transport is uniquely and selectively diminished in chronic lymphocytic leukemia B lymphocytes: the maximal velocity of transport is 10% of normal B lymphocytes. We examined L-system transport in chronic leukemic B lymphocytes after incubation with tetradecanoyl phorbol acetate to determine if the transport abnormality can be corrected by the apparent cell maturation induced by this agent. Amino acid uptake was measured using 2-amino-2-carboxy-bicycloheptane, an L-system specific synthetic amino acid. Marked enhancement of L-system transport occurred in each of 12 leukemic cell populations; the initial velocity of transport in phorbol ester-treated cells increased 8-fold and 14-fold at 16 and 40 h, respectively, compared with untreated cells. The Vmax of the L-system in phorbol ester-treated leukemic cells was similar to that of phorbol ester-treated normal B lymphocytes. The L-system enhancement of the leukemic cells paralleled the development of plasmacytoid features at 40 h. Uptake of leucine, a naturally occurring L-system amino acid, was also increased by tetradecanoyl phorbol acetate. Cycloheximide, 100 micrograms/ml, which inhibited over 90% of protein synthesis in phorbol ester-treated chronic leukemic cells, blocked completely the phorbol ester-induced L-system enhancement. Phorbol ester treatment restores the selective L-system transport defect in chronic lymphocytic leukemia B lymphocytes, and this process coincides with in vitro maturation of the leukemic cells.

Alpha-aminoisobutyric acid transport in human leukemic lymphocytes: in vitro characteristics and inhibition by cortisol and cycloheximide.

We have studied the transport of alpha-aminoisobutyric acid (AIB)-3-(14)C and its response to cortisol and cycloheximide in vitro in blood lymphocytes from untreated patients with chronic lymphocytic leukemia. The accumulation of AIB-3-(14)C increased in a linear fashion for 60 min, and reached an apparent steady state in 120 min. The initial rate of AIB accumulation (V(o)) varied from 1.1 to 10.2 mumoles/kg cell H(2)O per min in cells from 16 different patients; however, V(o) was reproducible in cells from five of six patients which were studied repeatedly over 1-9 months, and correlated positively with the lymphocyte count (r = 0.51, P = < 0.01). Virtually total inhibition of protein synthesis with cycloheximide was found to decrease the accumulation of AIB in cells from four patients which had high rates of AIB transport, but had no effect on transport in cells from four patients which accumulated AIB more slowly. These results indicate that active transport depends, in part, upon the presence of labile protein with a turnover rate which varies among different cell populations. Treatment with 10 muM cortisol for 240 min in vitro reduced the initial rate of AIB-3-(14)C accumulation (V(o)) by 43.4+/-4.1% (SE) (range, 9-66%) in cells from 16 patients. The degree of inhibition did not vary appreciably over a 9 month period in four of five patients. The effect of cortisol was proportional to its starting concentration, and developed at low concentrations (0.1-1.0 muM). Cortisol appears to decrease AIB accumulation by inhibiting active uptake, since it neither enhanced the exodus of AIB, nor inhibited apparently nonsaturable transport. Inhibition was noncompetitive in type, suggesting that cortisol decreases the total capacity of the active transport mechanism.Cortisol inhibited AIB transport indirectly by a process which involved de novo protein synthesis, since inhibition (a) appeared only after 60 min of treatment, (b) was present in treated cells which were subsequently incubated for 60 min in cortisol-free medium, and (c) failed to develop during simultaneous blockade of protein synthesis with cycloheximide, even when cycloheximide itself did not decrease AIB transport.

The relationships between arterial oxygen flow rate, oxygen binding by hemoglobin, and oxygen utilization after myocardial infarction.

The interrelationships of arterial oxygen flow rate index, oxygen binding by hemoglobin, and oxygen consumption have been examined in patients with acute myocardial infarction. Proportional extraction of oxygen increased in close association with decreasing oxygen flow rate, and hence, whole body oxygen consumption was constant over nearly a three-fold variation in arterial oxygen flow rate. A reduction in hemoglobin-oxygen affinity at in vivo conditions of pH. Pco(2) and temperature also occurred in proportion to the reduction in arterial oxygen flow rate. Therefore, the increased proportional removal of oxygen from arterial blood at low oxygen flow rates, required to maintain oxygen consumption, may have been facilitated by the reduced affinity of hemoglobin for oxygen at in vivo conditions. However, the decrease in affinity did not appear to explain more than 30-40% of the increased extraction. Respiratory alkalosis was a frequent occurrence in these patients and 2,3-diphosphoglycerate was positively associated with blood pH as well as with the time-averaged proportion of deoxyhemoglobin in arterial and venous blood.Hemoglobin-oxygen affinity measured at standard conditions and the mixed venous oxygen saturation were equally good indicators of reduced arterial oxygen flow rate in patients without shock. However, Svo(2) is more easily measured and is a more useful indicator of reduced oxygen flow rate, since its relationship to oxygen flow appears to be independent of affinity changes and time.

Maturation-associated changes in the peripheral cytoplasm of human neutrophils: a review.

The process of hemopoietic stem cell differentiation and proliferation leads to a large number of precursor cells committed to the neutrophil lineage. These precursor cells undergo a further limited degree of proliferation but, most importantly, undergo a dramatic process of maturation which alters the phenotype of the cell from a myeloblast, which is incapable of normal circulation and function into a segmented neutrophil capable of chemokinesis, chemotaxis, particle ingestion, microbicidal action, and other functions required to subserve the inflammatory process. This review describes the changes in the cell surface and cell cytoplasm that occur during precursor cell maturation and, to the extent possible, correlates molecular and macromolecular changes during maturation with the development of functional capacity.

Survival of granulocytic progenitors in the nonadherent and adherent compartments of human long-term marrow cultures.

We have studied the survival of granulocytic and monocytic progenitor cells (CFU-GM) in human liquid marrow cultures. CFU-GM were present in the nonadherent cell population for a mean of 12 weeks without recharging . Histochemical analysis of agar gels revealed that most day-7 colonies were of neutrophilic type (CFU-N), whereas the majority of day-14 colonies were of mixed neutrophilic-macrophagic type (CFU-NM) for the first four weeks of culture and became predominantly of macrophagic type (CFU-M) thereafter. Eosinophilic colonies (CFU-Eo) declined after week 2 of culture. We have documented that CFU-GM were present in the adherent layer of these cultures, and that the CFU-GM in the nonadherent compartment arise from the adherent layer. In addition, we have compared the pre-CFU-GM survival in the adherent and nonadherent populations and determined that these progenitors were rapidly depleted from both compartments though their survival at the end of week 1 was better in the adherent than in the nonadherent layer.

Plasma membrane vesicles prepared from unadhered monocytes: characterization of calcium transport and the calcium ATPase.

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.

Spatial genome organization during T-cell differentiation.

The spatial organization of genomes within the mammalian cell nucleus is non-random. The functional relevance of spatial genome organization might be in influencing gene expression programs as cells undergo changes during development and differentiation. To gain insight into the plasticity of genomes in space and time and to correlate the activity of specific genes with their nuclear position, we systematically analyzed the spatial genome organization in differentiating mouse T-cells. We find significant global reorganization of centromeres, chromosomes and gene loci during the differentiation process. Centromeres were repositioned from a preferentially internal distribution in undifferentiated cells to a preferentially peripheral position in differentiated CD4+ and CD8+ cells. Chromosome 6, containing the differentially expressed T-cell markers CD4 and CD8, underwent differential changes in position depending on whether cells differentiated into CD4+ or CD8+ thymocytes. Similarly, the two marker loci CD4 and CD8 showed distinct behavior in their position relative to the chromosome 6 centromere at various stages of differentiation. Our results demonstrate that significant spatial genome reorganization occurs during differentiation and indicate that the relationship between dynamic genome topology and single gene regulation is highly complex.