Sympathetic predominance before tourniquet deflation is associated with a reduction in arterial blood pressure after tourniquet deflation during total knee arthroplasty.

High dependency of arterial blood pressure (ABP) on enhanced sympathetic activity, which maintains vascular tone, leads to hypotension after hemodynamic insults that blunt the sympathetic activity. Therefore, we hypothesized that sympathovagal balance before tourniquet deflation (TD) determines the extent of a reduction in ABP after TD during total knee arthroplasty (TKA). Fifty-four hypertensive female patients undergoing TKA under spinal anesthesia were analyzed. The sympathovagal balance [low-to-high frequency ratio of heart rate variability (LF/HF)] before TD was defined as (LF/HF during 5 min before TD-preanesthetic LF/HF)/preanesthetic LF/HF (%). An increase in its value represents a shift in sympathovagal balance toward sympathetic predominance. The percent change in the mean ABP (MAP) after TD was defined as (minimum MAP during 10 min after TD-averaged MAP during 5 min before TD)/averaged MAP during 5 min before TD (%). Simple linear regression was performed to assess the correlation between the sympathovagal balance before TD and change in MAP after TD. The correlation was also assessed by multiple linear regression controlling for age, duration of tourniquet inflation, and spinal anesthesia-induced hypotension. Thirty-two minutes (on average) after tourniquet inflation, the MAP was decreased by 12.1 (-3.0 to 47.9) % [mean (range)] upon TD (P<0.001). The sympathovagal balance before TD was negatively proportional to the change in MAP after TD in both simple and multiple linear regression models (R2=0.323 and 0.340, P<0.001). A shift in sympathovagal balance toward sympathetic predominance before TD is associated with a decrease in ABP after TD.

Suppression of creep-regime dynamics in epitaxial ferroelectric BiFeO3 films.

Switching dynamics of ferroelectric materials are governed by the response of domain walls to applied electric field. In epitaxial ferroelectric films, thermally-activated 'creep' motion plays a significant role in domain wall dynamics, and accordingly, detailed understanding of the system's switching properties requires that this creep motion be taken into account. Despite this importance, few studies have investigated creep motion in ferroelectric films under ac-driven force. Here, we explore ac hysteretic dynamics in epitaxial BiFeO3 thin films, through ferroelectric hysteresis measurements, and stroboscopic piezoresponse force microscopy. We reveal that identically-fabricated BiFeO3 films on SrRuO3 or La0.67Sr0.33MnO3 bottom electrodes exhibit markedly different switching behaviour, with BiFeO3/SrRuO3 presenting essentially creep-free dynamics. This unprecedented result arises from the distinctive spatial inhomogeneities of the internal fields, these being influenced by the bottom electrode's surface morphology. Our findings further highlight the importance of controlling interface and defect characteristics, to engineer ferroelectric devices with optimised performance.

The effect of human fetal liver-derived mesenchymal stem cells on CD34+ hematopoietic stem cell repopulation in NOD/Shi-scid/IL-2Rã(null) mice.

Mesenchymal stem cells (MSCs) are progenitors that are capable of differentiating into mesenchymal tissues. They are known to support allogeneic hematopoietic stem cell transplantation by facilitating engraftment without increasing the risk of graft-versus-host disease. We optimized culture conditions for human fetal liver-derived MSCs (hFL-MSCs) to investigate the role of hFL-MSCs on repopulation of hematopoietic stem cells in NOD/Shi-scid/IL-2Rγ(null) (NOG) mice using CD34(+) hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB). FL-MSCs and CD34(+) HSCs were prepared from fetal liver and UCB, respectively. Twenty-four hours after irradiation, CD34(+) HSCs and hFL-MSCs were injected intravenously and intratibially into NOG mice. During 24 weeks posttransplantation, engraftment levels of human cells were analyzed in bone marrow, peripheral blood, and spleen of transplanted mice by flow cytometry. hFL-MSCs showed a fibroblast-like morphology and immunophenotypic characteristics appropriate for MSCs. hFL-MSCs prolonged the survival of NOG mice that had been cotransplanted with UCB CD34(+) cells. Fluorescence-activated cell-sorting analysis showed that engraftment of human cells was increased by cotransplantation of hFL-MSCs. However, significant enhancement of human cell engraftment was not detected in NOG mice regardless of the number of cotransplanted MSCs. Although survival of repopulating NOG mice and engraftment of human cells were prolonged by cotransplantation of hFL-MSCs, 8.0 × 10(6) MSCs were not sufficient to increase HSC engraftment in irradiated NOG mice in vivo.

Associations of fat mass and lean mass with bone mineral density differ by levels of persistent organic pollutants: National Health and Nutrition Examination Survey 1999-2004.

Persistent organic pollutants (POPs), xenobiotics that accumulate in fat tissue, may impair bone metabolism. We studied (1) the association of bone mineral density (BMD) with POPs and (2) whether associations of fat mass (FM) or lean mass (LM), two components of body composition, with BMD differed depending on levels of POPs. Participants aged ≥ 20 in the National Health and Nutrition Examination Survey 1999-2004 were included (n=2769). Eight POPs with detection rate ≥ 80% and three skeletal subregions (left arm, pelvis, and right leg) were selected. All analyses were stratified by gender and age (cutpoint 50 years or more). POPs at background concentrations were mostly unassociated with BMD. However, the associations of FM and LM with BMD depended on POPs concentrations, in particular with BMD of the left arm (usually not weight-bearing) in postmenopausal women. When POPs concentrations were low, FM showed inverse associations with BMD while LM showed positive associations. However, when POPs levels were high, FM showed positive associations with BMD while the positive associations between LM and BMD weakened. POPs may biologically modify the associations of FM and LM with BMD, especially among postmenopausal women, possibly explaining inconsistent associations between FM and BMD in previous epidemiological studies.

Altered calcium dynamics mediates P19-derived neuron-like cell responses to millimeter-wave radiation.

Intracellular calcium oscillations have long been recognized as a principal mediator of many vital cellular activities. Furthermore, Ca(2+) dynamics can be modulated by external physical cues, including electromagnetic fields. While cellular responses to low-frequency electric fields have been established, the possible non-thermal effects of millimeter-wave (MMW) radiation are still a subject of discussion and debate. We used mouse embryonic stem cell-derived neuronal cells and a custom-built 94 GHz applicator to examine in real time the altered Ca(2+) oscillations associated with MMW stimulation. MMW irradiation at 18.6 kW/m(2) nominal power density significantly increased the Ca(2+) spiking frequency in the cells exhibiting Ca(2+) activity. The N-type calcium channels, phospholipase C enzyme, and actin cytoskeleton appear to be involved in mediating increased Ca(2+) spiking. Reorganization of the actin microfilaments by a 94 GHz field seems to play a crucial role in modulating not only Ca(2+) activity but also cell biomechanics. Many but not all observed cellular responses to MMW were similar to thermally induced effects. For example, cell exposure to a 94 GHz field induced nitric oxide production in some morphologically distinct neuronal cells that could not be reproduced by thermal heating of the cells up to 42 degrees C. The highest observed average temperature rise in the MMW exposure chamber was approximately 8 degrees C above the room temperature, with possible complex non-uniform microscopic distribution of heating rates at the cell level. Our findings may be useful to establish quantitative molecular benchmarks for elucidation of nociception mechanisms and evaluation of potential adverse bioeffects associated with MMW exposure. Moreover, control of Ca(2+) dynamics by MMW stimulation may offer new tools for regulation of Ca(2+)-dependent cellular and molecular activities, for example, in tissue engineering applications.

Nonthermal effects of radiofrequency-field exposure on calcium dynamics in stem cell-derived neuronal cells: elucidation of calcium pathways.

Intracellular Ca(2+) spikes trigger cell proliferation, differentiation and cytoskeletal reorganization. In addition to Ca(2+) spiking that can be initiated by a ligand binding to its receptor, exposure to electromagnetic stimuli has also been shown to alter Ca(2+) dynamics. Using neuronal cells differentiated from a mouse embryonic stem cell line and a custom-built, frequency-tunable applicator, we examined in real time the altered Ca(2+) dynamics and observed increases in the cytosolic Ca(2+) in response to nonthermal radiofrequency (RF)-radiation exposure of cells from 700 to 1100 MHz. While about 60% of control cells (not exposed to RF radiation) were observed to exhibit about five spontaneous Ca(2+) spikes per cell in 60 min, exposure of cells to an 800 MHz, 0.5 W/kg RF radiation, for example, significantly increased the number of Ca(2+) spikes to 15.7+/-0.8 (P<0.05). The increase in the Ca(2+) spiking activities was dependent on the frequency but not on the SAR between 0.5 to 5 W/kg. Using pharmacological agents, it was found that both the N-type Ca(2+) channels and phospholipase C enzymes appear to be involved in mediating increased Ca(2+) spiking. Interestingly, microfilament disruption also prevented the Ca(2+) spikes. Regulation of Ca(2+) dynamics by external physical stimulation such as RF radiation may provide a noninvasive and useful tool for modulating the Ca(2+)-dependent cellular and molecular activities of cells seeded in a 3D environment for which only a few techniques are currently available to influence the cells.

Morphology and adhesion of mesenchymal stem cells on PLLA, apatite and apatite/collagen surfaces.

Biomimetic apatite/collagen composite coating, previously reported particularly with regard to its fabrication, characterization and interaction with osteoblast-like cells, has been investigated in this study to understand the response of human mesenchymal stem cells (hMSC) to such surface. PLLA films and PLLA films with apatite coating were compared with PLLA films with apatite/collagen composite coating. The hMSC morphology in response to such conditions was first observed using fluorescence microscopy. To further understand such cell-material interactions at a molecular level, integrin expression, actin assembly and vinculin-positive focal adhesion plaques were examined. Our results demonstrated that spreading of stem cells on the apatite/collagen composite surface was determined best among the three types of surfaces, followed by the apatite surface and then the PLLA control. Integrin expression on the apatite/collagen surface was higher than those on the apatite surface and PLLA surface. Immunostaining for vinculin and actin suggested that the composite coating on PLLA enhanced the formation of focal adhesion.

Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein-tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1DeltaRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.

Integrin-dependent human macrophage migration induced by oscillatory electrical stimulation.

Electrical stimulation has been used to promote wound healing. The mechanisms by which such stimulation could interact with biological systems to accelerate healing have not been elucidated. One potential mechanism could involve stimulation of macrophage migration to the site of a wound. Here we report that oscillatory electric fields induce human macrophage migration. Macrophages exposed to a 1 Hz, 2 V/cm field show an induced migration velocity of 5.2+/-0.4 x 10(-2) microm/min and a random motility coefficient of 4.8+/-1.4 x 10(-2) microm2/min on a glass substrate. Electric field exposure induces reorganization of microfilaments from ring-like structures at the cell periphery to podosomes that are confined to the contact sites between cell and substrate, suggesting that the cells are crawling on glass. Treatment of cells with monoclonal antibodies directed against beta2-integrins prior to field exposure prevents cell migration, indicating that integrin-dependent signaling pathways are involved. Electric fields cause macrophage migration on laminin or fibronectin coated substrates without inducing podosome formation or changes in cellular morphology. The migration velocity is not significantly altered but the random movement is suppressed, suggesting that cell movements on a laminin- or fibronectin-coated surface are not mediated by cell crawling. It is suggested that electric field-induced macrophage migration utilizes several modes of cell movement, including cell crawling and possibly cell rolling.

Ultrastructure of the Z-organ and Parts of the Female Genital Tract in Xiphinema coxi coxi.

Ultrastructure of the Z-organ and associated apophyses in Xiphinema coxi coxi was studied by transmission electron microscopy to determine their structural origin and relationship with other parts of the genital tract. The Z-organ of X. coxi coxi is oval-shaped, ca. 30 microm long and 16 microm wide. It is clearly distinguished from the other parts of the female genital tract by its thick muscular outer wall, epithelium-lined lumen, and 4-5 centrally located apophyses. Each apophysis is continuous with the epithelial lining of the Z-organ, suggesting that it originated from epithelium. The apophyses appear as thickened and densely folded masses forming numerous interlaced pores and (or) chambers containing mucous-like materials and electron-dense crystals. These apophyses are characteristic of a typical Z-organ; no globular structures characteristics of the pseudo-Z-organ were observed. The thickness of the muscular layer of the oviduct and uterus varied with position. The overall Z-organ ultrastructure of this study, including body wall and internal apophyses, was comparable to the typical Z-organ of X. ifacolum. This suggests that X. coxi coxi should be classified as a Xiphinema species that contains the typical Z-organ.

Mild spherocytosis and altered red cell ion transport in protein 4. 2-null mice.

Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4. 2-null (4.2(-/-)) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2(-/-) RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4. 2(-/-) RBCs are normal. Band 3 and band 3-mediated anion transport are decreased. Protein 4.2(-/-) RBCs show altered cation content (increased K+/decreased Na+)resulting in dehydration. The passive Na+ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4. 2(-/-) RBCs are significantly increased. Protein 4.2(-/-) RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2(-/-) RBCs compared with controls. The increased passive Na+ permeability of 4.2(-/-) RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.

Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase.

Although estrogen is known to stimulate nitric oxide synthesis in vascular endothelium, the molecular mechanisms responsible for this effect remain to be elucidated. Using quantitative immunofluorescence imaging approaches, we have investigated the effect of estradiol on the subcellular targeting of endothelial nitric oxide synthase (eNOS) in bovine aortic endothelial cells. In unstimulated endothelial cells, eNOS is predominantly localized at the cell membrane. Within 5 min after the addition of estradiol, most of the eNOS translocates from the membrane to intracellular sites close to the nucleus. On more prolonged exposure to estradiol, most of the eNOS returns to the membrane. This effect of estradiol is evident at a concentration of 1 pM, and a maximal estradiol effect is seen at a concentration of 1 nM. Neither progesterone nor testosterone has any effect on eNOS distribution. After estradiol addition, a transient rise in intracellular Ca2+ concentration precedes eNOS translocation. Both the Ca2+-mobilizing and eNOS-translocating effects of estradiol are completely blocked by the estrogen receptor antagonist ICI 182,780, and the intracellular Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevents estradiol-induced eNOS translocation. Use of the nitric oxide-specific dye diaminofluorescein shows that estradiol treatment increases nitric oxide generation by endothelial cells; this response is blocked by ICI 182,780 and by the eNOS inhibitor Nomega-nitro-L-arginine. These results show that estradiol induces subcellular translocation of eNOS by a rapid, Ca2+-dependent, receptor-mediated mechanism, and they suggest a nongenomic role for estrogen in the modulation of NO-dependent vascular tone.

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.

Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells.

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-alphaAQP1) in the undeformed red cell membrane is 66 +/- 10% and the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm2/s. F-alphaAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-alphaAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, approximately 50% of the aspirated F-alphaAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-alphaAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.

Receptor-regulated translocation of endothelial nitric-oxide synthase.

The endothelial nitric-oxide synthase (eNOS) is activated by transient increases in intracellular Ca2+ elicited by stimulation of diverse receptors, including bradykinin B2 receptors on endothelial cells. eNOS and B2 receptors are targeted to specialized signal-transducing domains in the plasma membrane termed plasmalemmal caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation, but in resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. We used a quantitative approach exploiting immunofluorescence microscopy to investigate regulation of the subcellular distribution of eNOS in endothelial cells by bradykinin and Ca2+. In resting cells, most of the eNOS is localized at the cell membrane. However, within 5 min following addition of bradykinin, nearly all the eNOS translocates to structures in the cell cytosol; following more protracted incubations with bradykinin, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The bradykinin-induced internalization of eNOS is completely abrogated by the intracellular Ca2+ chelator BAPTA; conversely, Ca2+-mobilizing drugs and agonists promote eNOS translocation. These results establish that eNOS targeting to the membrane is labile and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as preformed stable species.

Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.

Regulation of band 3 rotational mobility by ankyrin in intact human red cells.

Ankyrin mutations and combined spectrin and ankyrin deficiency are prominent features of red blood cells (RBCs) in patients with hereditary spherocytosis (HS). Band 3 is the most abundant integral protein in the human RBC membrane. Previous studies have shown that the lateral mobility, but not the rotational mobility, of band 3 is increased in RBCs from patients with severe autosomal recessive HS and selective spectrin deficiency. These observations are consistent with the steric hindrance model of lateral mobility restriction. Here we use the fluorescence photobleaching recovery and polarized fluorescence depletion techniques to measure the lateral and rotational mobility of band 3 in intact RBCs from six patients with HS, ankyrin mutations, and combined spectrin and ankyrin deficiency. As predicted by the steric hindrance model, the lateral diffusion rate of band 3 is greater in spectrin- and ankyrin-deficient RBCs than in control cells, and the magnitude of the increase correlates with the degree of spectrin deficiency. Unlike RBCs from patients with HS and selective spectrin deficiency, however, HS RBCs with ankyrin mutations exhibit a marked increase in band 3 rotational diffusion. The magnitude of the increase correlates inversely with the ankyrin/band 3 ratio and with the fraction of band 3 retained in the membrane skeleton following detergent extraction. These data suggest that ankyrin deficiency relaxes rotational constraints on the major (slowly rotating) population of band 3 molecules. Increases in band 3 rotation could be due to release of band 3 from low-affinity binding sites on ankyrin.

Red cell membranes of ankyrin-deficient nb/nb mice lack band 3 tetramers but contain normal membrane skeletons.

The role of ankyrin in the formation and stabilization of the spectrin-based skeletal meshwork and of band 3 oligomers was studied by characterizing, in nb/nb mouse red cells, the effect of ankyrin deficiency on skeletal ultrastructure, band 3-skeleton associations, and band 3 oligomeric states. Despite severe ankyrin deficiency, nb/nb mouse red cell skeletal components formed a relatively uniform two-dimensional hexagonal array of junctional complexes cross-linked by spectrin tetramers. Treatment of nb/nb ghosts with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether) resulted in nearly complete extraction of band 3. The extracted band 3 was present exclusively as band 3 dimers. Fluorescence photobleaching recovery and polarized fluorescence depletion measurements showed increases in the laterally (33% vs 10%) and rotationally (90% vs 76%) mobile fractions of band 3 in intact nb/nb compared to control red cells. The rotational correlation time of the major fraction of band 3 molecules was 10-fold shorter in nb/nb compared to control red cells, indicating a significant relaxation of rotational constraints in nb/nb cells. These data suggest that, although ankyrin plays a major role in strengthening the attachment of the skeleton to the membrane bilayer, ankyrin is not required for the formation of a stable two-dimensional spectrin-based skeleton. The absence of band 3 tetramers in the membrane of ankyrin-deficient red cells suggests that ankyrin is required for the formation of stable band 3 tetramers.

A multiparameter analysis of sickle erythrocytes in patients undergoing hydroxyurea therapy.

During 24 weeks of hydroxyurea treatment, we monitored red blood cell (RBC) parameters in three patients with sickle cell disease, including F-cell and F-reticulocyte profiles, distributions of delay times for intracellular polymerization, sickle erythrocyte adherence to human umbilical vein endothelial cells in a laminar flow chamber, RBC phthalate density profiles, mean corpuscular hemoglobin concentration and cation content, reticulocyte mean corpuscular hemoglobin concentration, 1H-nuclear magnetic resonance transverse relaxation rates of packed RBCs, and plasma membrane lateral and rotational mobilities of band 3 and glycophorins. Hydroxyurea increases the fraction of cells with sufficiently long delay times to escape the microcirculation before polymerization begins. Furthermore, high pretreatment adherence to human umbilical vein endothelial cells of sickle RBCs decreased to normal after only 2 weeks of hydroxyurea treatment, preceding the increase in fetal hemoglobin levels. The lower adhesion of sickle RBCs to endothelium would facilitate escape from the microcirculation before polymerization begins. Hydroxyurea shifted several biochemical and biophysical parameters of sickle erythrocytes toward values observed with hemoglobin SC disease, suggesting that hydroxyurea moderates sickle cell disease toward the milder, but still clinically significant, hemoglobin SC disease. The 50% reduction in sickle crises documented in the Multicenter Study of Hydroxyurea in Sickle Cell Disease is consistent with this degree of erythrocyte improvement.

Reorganization of microfilament structure induced by ac electric fields.

AC electric fields induce redistribution of integral membrane proteins. Cell-surface receptor redistribution does not consistently follow electric field lines and depends critically on the frequency of the applied ac electric fields, suggesting that mechanisms other than electroosmosis are involved. We hypothesized that cytoskeletal reorganization is responsible for electric field-induced cell-surface receptor redistribution, and used fluorescence video microscopy to study the reorganization of microfilaments in human hepatoma (Hep3B) cells exposed to low-frequency electric fields ranging in strength from 25 mV/cm to 20 V/cm (peak to peak). The frequency of the applied electric field was varied from 1 to 120 Hz and the field exposure duration from 1 to 60 min. In control cells, cytoplasmic microfilaments were aligned in the form of continuous parallel cables along the longitudinal axis of the cell. Exposure of cells to ac electric fields induced alterations in microfilament structure in a manner that depended on the frequency of the applied field. A 1 or 10 Hz ac field caused microfilament reorganization from continuous, aligned cable structures to discontinuous globular patches. In contrast, the structure of microfilaments in cells exposed to 20-120 Hz electric fields did not differ from that in control cells. The extent of microfilament reorganization increased nonlinearly with the electric field strength. The characteristic time for microfilament reorganization in cells exposed to a 1 Hz, 20 V/cm electric field was approximately 5 min. Applied ac electric fields could initiate signal transduction cascades, which in turn cause reorganization of cytoskeletal structures.