Further characterization of the effects of alpha-1-acid glycoprotein on the passage of human erythrocytes through micropores

Effects of human alpha-1-acid glycoprotein (AG) on the passage of human red blood cell(s) (RBC) through membrane filters with micropores were examined in vitro. RBCs, with a mean major diameter of 7.2 micron, that had been suspended at 1% in physiological phosphate-buffered saline (PBS), were filtered through membrane filters of various pore diameters under positive pressure. The percentages of cells that passed through the micropores and of cells hemolyzed during filtration were determined. RBCs suspended in PBS did not pass through micropores that had an average pore diameter of 3 micron; instead hemolysis took place. Neither temperature nor applied pressure affected cell passage; but when AG at 0.1 mg/ml or above was added to an RBC-suspension, it promoted cell passage through the 3 micron micropores and reduced the degree of hemolysis. The effects of AG were dose dependent up to a concentration of 0.5 mg/ml. The addition of AG to an RBC-suspension that contained 90% human serum had the same additive effects. Washing AG-treated RBCs with normal saline produced a marked decrease in cell passage through the 3 micron pores. Fluorescence antibody staining revealed that the exogenous AG was localized on the membrane surface of the RBCs. Our results suggest that the AG bound to the surface of the RBCs acts as a lubricant between the RBCs and the wall of the micropore; this would facilitate RBC-passage through the micropores.     

Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.     

Accurate determination of mean cell volume by isotope dilution in erythrocyte populations with variable deformability.

Variations in erythrocyte deformability and morphology lead to artifacts in electronic determinations of mean cellular volume (MCV) by the aperture-impedance method. The micropipette-aspiration technique loses accuracy when applied to severely aberrant cells such as dense sickle cells. A new light-scattering technique requires that the cells be capable of undergoing isovolumetric sphering. In contrast, the isotope-dilution (ID) method measures absolute mean volume and is free of artifacts associated with abnormal deformability or morphology. It does not depend on any algorithms or correction factors and does not subject the cells to any stringent processing, not even centrifugation. The ID method can be used to determine the mean volume of red cells in hypo- or hypertonic media or in the presence of pharmacologic agents. It requires no more than a 1-ml aliquot of suspended cells at a hematocrit of at least 30%. The cells can be readily recovered, washed, and reused. Using EDTA labeled with 57Co as an extracellular space marker we have used ID to determine the MCV of fractionated normal human red blood cells (RBC), unfractionated RBC containing SS hemoglobin, and RBC from four other mammalian species. In the case of human RBC obtained from eight normal donors, we obtained mean MCV values (+/- SD) of 83.6 +/- 3.0, 87.5 +/- 3.9, and 76.5 +/- 5.3 fl for unfractionated and top and bottom 10% density fractions, respectively. The value 83.6 is significantly lower than the generally accepted range of 89-91 indicated by electronic analyzers calibrated against spun microhematocrits. The discrepancy of about 7% can account for the difference between mean cell hemoglobin concentration (MCHC) data determined by a calibrated Coulter Counter and corresponding data obtained with paired samples using a cyanmethemoglobin procedure specified in NCCLS Standard H15-A and corrected for trapped plasma.     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

Aggregation behavior and electrophoretic mobility of red blood cells in various mammalian species

Differences of red blood cell (RBC) aggregation among various mammalian species has been previously reported for whole blood, for RBC in autologous plasma, and for washed RBC re-suspended in polymer solutions. The latter observation implies the role of cellular factors, yet comparative studies of such factors are relatively limited. The present study thus investigated RBC aggregation and RBC electrophoretic mobility (EPM) for guinea pigs, rabbits, rats, humans and horses; RBC were re-suspended in isotonic 500 kDa dextran solutions for the EPM and aggregation measurements, with aggregation studies also done in autologous plasma. Salient results included: (1) species-specific RBC aggregation in both plasma and dextran (horse > human > rat > rabbit approximately = guinea pig) with a significant correlation between aggregation in the two media; (2) similar EPM values in PBS for rat, human and horse, a lower value for guinea pig, and a markedly reduced EPM for rabbit RBC; (3) EPM values in dextran with a rank order identical to that for cells in PBS; (4) relative EPM results indicating formation of a polymer-poor, low viscosity depletion layer at the RBC surface (greatest depletion for horse RBC). EPM-aggregation correlations were evident and generally consistent with the Depletion Model for aggregation, yet did not fully explain differences between species; additional studies at various ionic strengths and with various dextran fractions thus seem warranted.     

Blood volume in inbred strain BALB/c, CBA/J and C57BL/10 mice determined by means of 59Fe-labelled red cells and 59Fe bound to transferrin.

Circulating red blood cell (RBC) and plasma volume was determined in male inbred strain BALB/c, CBA/J and C57BL/10 mice by parallel use of the 59Fe-labelled RBC dilution and the dilution of 59Fe bound to transferrin. The whole blood volumes values derived from the venous haematocrit and plasma volume were about double the values calculated from the venous haematocrit and circulating RBC volume. Comparison of the two methods thus explains the marked differences in different studies of blood volume in mice and shows that correct values can be obtained only by parallel measurements of RBC and plasma volume by separate methods, or by correcting the venous haematocrit to whole body haematocrit. Combination of the labelled RBC method and the 59Fe-transferrin method showed the blood volume values in the above strains of mice to be 10.35 +/- 0.16, 7.32 +/- 0.10 and 7.94 +/- 0.15 ml/g b.w. respectively. The ratio of whole body to venous haematocrit in these strains was was 57.3 +/- 1.6%, 68.0 +/- 1.8% and 69.5 +/- 2.2%. Significant interstrain differences were demonstrated in RBC, plasma and blood volume and in the venous and whole body haematocrit and their ratio.     

Alteration of red cell membrane viscoelasticity by heat treatment: effect on cell deformability and suspension viscosity

The membrane shear elastic modulus (mu) and the time constant for extensional shape recovery (tc) were measured for normal, control human red blood cells (RBC) and for RBC heat treated (HT) at 48 degrees C. Three separate methods for the measurement of mu were compared (two used a micropipette and one employed a flow channel), and the membrane viscosity (n) was calculated from the relation n = mu. tc. The deformability of HT and control cells was evaluated using micropipette techniques, and the bulk viscosity of RBC suspensions at 40% hematocrit was measured. The shear elastic modulus, or "membrane rigidity", was more than doubled by heat treatment, although both the absolute value for mu and the estimate of the increase induced by heat treatment varied depending on the method of measurement. Heat treatment caused smaller increases in membrane viscosity and in membrane bending resistance, and only minimal changes in cell geometry. The deformability of HT cells was reduced: 1) the pressure required for cell entry (Pe) into 3 micrometers pipettes was increased, on average, by 170%; 2) at an aspiration pressure (Pa) exceeding Pe, longer times were required for cell entry into the same pipettes. However, when Pa was scaled relative to the mean entry pressure for a given sample (i.e, Pa/Pe), entry times were similar for control and HT cells. Bulk viscosity of HT RBC suspensions was elevated by approximately 12% on average (shear rates 75 to 1500 inverse seconds). These findings suggest that alteration of RBC membrane mechanical properties, similar to those induced by heat treatment, would most affect the in vivo circulation in regions where vessel dimensions are smaller than cellular diameters.     

Effects of pressure on red blood cell geometry during micropipette aspiration

Micropipette aspiration is a potentially useful and accurate technique to measure red blood cell (RBC) geometry. Individual RBCs are partially aspirated and from the resulting sphere diameter, total cell length, and pipette diameter, membrane area and cell volume can be calculated. In this study we have focused on possible shape artifacts associated with the aspirated portion of RBC. We observed that the apparent RBC geometry (calculated area and volume) changed markedly (P < 0.001) with the applied aspiration pressure; for normal human RBC the area increased by 5.6 +/- 0.6% and volume decreased by 4.7 +/- 0.6% when the aspiration pressure was increased from 20 to 100 mm water. The calculated membrane area dilation modulus was 7.4 dyn/ cm, which is far below the expected value, and microscopic observations revealed a membrane folding artifact as a possible artifact. These assumptions were strengthened by using a short-duration (3 s) pressure peak of 20-100-20 mm water. The folding then disappeared permanently, but a small (0.31 +/- 0.09%; P < 0.001) area decrease was detected which yields a realistic dilation modulus of 215 dyn/cm. We conclude that membrane folding can critically affect RBC micropipette measurements and that a transient pressure peak can unfold the RBC membrane, thus allowing accurate measurements of RBC geometry.     

Effect of procaine hydrochloride on the aggregation behavior and suspension viscoelasticity of human red blood cells

The present study examined the effects of procaine hydrochloride (PRHCL), a cationic local anesthetic, on the aggregation behavior of human red blood cells (RBC); the effects of PRHCL on RBC suspension viscoelasticity, cell shape, volume and density were also investigated. Four indices of RBC aggregation, induced by autologous plasma or 3 g% dextran T70, were evaluated by a computerized light transmission method, and the viscous and elastic components of the complex viscosity were determined by oscillatory viscometry. Low concentrations of PRHCL (8 x 10(-5) to 8 x 10(-4) M) significantly (p less than 0.05 or better) reduced the extent of aggregation (maximal decrease of 22% at 8 x 10(-4) M), but did not alter the viscoelastic components, cell shape, volume or density. The anti-aggregating effect of PRHCL (8 x 10(-4) M) in plasma significantly (p less than 0.005) decreased with time; this temporal effect was abolished by addition of eserine (1 x 10(-4) M). High concentrations of PRHCL (8 x 10(-2) M) caused: 1) increased extent of aggregation and decreased strength of the aggregates (p less than 0.01 or better); 2) elevation of both viscoelastic components for cells in plasma or buffer; 3) a discocyte-stomatocyte shape change; 4) decreased cell density (p less than 0.001) without alteration of cell volume. Our results at low concentrations of PRHCL suggest a mechanism based on an increase of RBC negative surface potential; at the highest concentration, the effects are most likely due to altered cell shape and deformability, and to decreased RBC negative surface potential.     

Optical evaluation of red blood cell geometry using micropipette aspiration.

Although red blood cell (RBC) geometry has been extensively studied by micropipette aspiration, the small size of RBC and pipettes vs. the optical resolution of light microscopy suggests the need to consider potential errors. The present study addressed such difficulties and investigated four specific problems: (1) use of exact equations to calculate RBC membrane area and volume; (2) calibration of the pipette internal diameter (PID); (3) correction for a noncylindrical pipette barrel; (4) diffraction distortion of the RBC image. The observed PID represents a cylinder lens enlargement that can be theoretically derived from the glass/buffer refractive index ratio (1.49/1.33 = 1.12). This enlargement was experimentally confirmed by: (1) studying pipettes bent to allow measurement through the barrel (horizontal) and at the orifice (vertical), with a resulting diameter ratio of 1.12 +/- 0.01; (2) and by replacing the surrounding buffer with immersion oil and hence abolishing the lens phenomenon (ratio = 1.12 +/- 0.02). In addition, use of aspirated oil droplets demonstrated a 3.2 +/- 0.2% error when the PID is focused at a sharp, maximum diameter. The average pipette cone angle was 1.49 +/- 0.09 degrees and varied considerably with pipette pulling procedures; calculated tongue geometry inside the pipette was affected by the noncylindrical pipette barrel. The RBC diffraction error, demonstrated by touching two aspirated cells held by opposing pipettes, was 0.091 +/- 0.002 microns. The PID, cone angle, and diffraction artifacts significantly (p < 0.001) affected calculated RBC geometry (average errors up to 5.4% for area and 9.6% for volume). Two new methods to calculate, rather than directly measure, the PID from images of a single RBC, during either osmotic or pressure manipulation, were evaluated; the osmotic method closely predicted the PID, whereas the pressure method markedly underestimated the PID. Our results thus confirm the need to consider the above-mentioned phenomena when determining RBC geometric parameters via micropipette aspiration.     

Comparative studies of LFA-1/ICAM-1 interaction by micropipette and flow chamber techniques

Interaction of lymphocyte function-associated antigen-1 (LFA-1) with intercellular adhesive molecule-1 (ICAM-1) is important in a number of cellular events, including inflammation, adhesion, transendothelial migration. The aim of this work was to study comparatively the adhesive interaction between LFA-1 and ICAM-1 by a micropipette technique and a flow chamber method, and also to explore the effects of tumor necrosis factor (TNF-alpha), phytohemagglutinin (PHA), and tetramethylpyrazine (TMP) on this interaction. The adhesion probability (Pa) between a lymphocyte cell line SKW-3 expressing LFA-1 and a red blood cell (RBC) coated with soluble ICAM-1 was approached by the micropipette technique, while the flow chamber allowed to observe the firm adhesion of SKW-3 on human umbilical vein endothelial cells (HUVECs). Experimental results show that PHA stimulation of lymphocytes resulted in significant increases in the adhesion probability (Pa) and in number of firmly adhered lymphocytes to HUVECs, but TMP treatment could significantly inhibit such increases.     

Water permeability of human hematopoietic stem cells

It is currently impossible to isolate or identify human hematopoietic progenitor cells from the bone marrow, yet the biophysical properties of these cells are important for the development of techniques to isolate and preserve stem cells for transplantation. Osmotic permeability properties of human bone marrow stem cells were estimated from the kinetics of cell damage in a hypotonic solution measured using in vitro colony assays for multipotential (CFU-GEMM) and committed (BFU-E, CFU-GM) progenitor cells. Cells exposed to a hypotonic solution swell as a result of water influx, and the rate of change of volume is proportional to the hydraulic conductivity of the plasma membrane. Cell damage occurs when the cell volume exceeds the maximum tolerable volume, so the hydraulic conductivity can be estimated from the kinetics of cell damage. For all the progenitor cells studied, the mean value of the hydraulic conductivity was 0.283 micron3/micron2/min/atm at 20 degrees C, with an Arrhenius activation energy of 6.41 kcal/mole. No significant differences were observed in the osmotic properties of the various progenitor cells. These data were used to predict the osmotic responses of human bone marrow stem cells at subzero temperatures during freezing.     

Quantitative ultrastructural study of nucleolus-organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis)

The quantitative characteristics of chromosomal nucleolus-organizing regions (NORs) and some other nucleolar components were studied on ultra-thin sections of pig embryo kidney cells (PK cells). It was shown that: 1) nucleoli-per-cell volumes were 3 times smaller in the G0 period than in the G2 period; 2) the number of fibrillar centers (FCs) per cell in the G0 period, the G2 period, and at metaphase was equal to 7, 33.7, and 8, respectively; 3) mean volumes of individual FCs in the G0 period (0.033 +/- 0.005 micron3), G2 period (0.014 +/- 0.001 micron3), and at metaphase (0.025 +/- 0.002 micron3) were significantly different; 4) the total volumes of FCs calculated per haploid set of chromosomes were practically the same in the G0 (0.105 micron3) and G2 (0.107 micron3) periods, but were twice as large as those at metaphase (0.04-0.05 micron3). These data show that partial activation and inactivation of ribosomal genes in interphase PK cells are not accompanied by a considerable change in the total volume of FCs and may be due to the fragmentation and fusion of individual FCs. Complete inactivation of ribosomal genes in mitosis results in a decrease of total volumes of FCs per cell; 5) in G0 and G2 periods the total volume of the dense fibrillar component per nucleolus is practically proportional to the nucleolus volume (r = 0.99); 6) in the G2 period, the nucleolus volume is also proportional to the number of FCs (r = 0.99; 7) the volume of the dense fibrillar component within individual fibrillar complexes is not a constant one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.     

Comparison of five methods for concentrating progenitor cells in human marrow transplantation

Enrichment of bone marrow (BM) aspirates is an important prerequisite prior to in vitro treatment or cryopreservation. In this regard, we have analyzed the results obtained on 190 BM processed by the following 5 techniques: HES sedimentation with centrifugation; COBE 2991 blood cell processor; Ficoll/hypaque (F/H) gradient centrifugation; Continuous flow cell separator (CS 3000 Fenwal); Semicontinuous blood cell separator (Dideco T 90). Each procedure was evaluated by measuring the recovery of nucleated marrow cells (NC), mononuclear cells (MNC), committed progenitor cells (CFU-GM), the reduction of BM volume and the removal of red blood cells (RBC) and polymorphonuclear cells (PMN). The results of this comparative study show that F/H gradient on a COBE 2991 cell washer provides the most efficient system for purifying a MNC fraction (89% recovery) from unwanted cells (RBC less than 2% and PMN less than 2%) in a very small volume (98% reduction) with a good recovery of CFU-GM (80%).     

Interleukin-6 deficiency increases blood volume without altering body composition in young mice

This study was carried out to measure blood volume in interleukin-6 (IL-6) deficient and the wild type mice and to relate any differences to concomitant changes in body composition. Young animals (8-20 weeks of age) were used in this study. The red blood cell (RBC) and plasma volumes were measured by isotope-dilution using Cr(51)-labeled erythrocytes and I(131)-labeled human albumin injected simultaneously in each animal. Red blood cells and plasma volumes and hence total measured blood volume were significantly larger in the IL-6-deficient than in either male or female wild type mice. There were no differences in either whole body hematocrit or Fcells ratio. There were no differences in water, fat, protein and mineral content between the genotypes of either gender. Thus, IL-6-deficient mice at young age have a significantly greater blood volume than the wild type mice without concomitant changes in body composition. We suggest that the changes in blood volume may reflect a role for IL-6 in the regulation of the peripheral circulation.     

Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.     

O6-3-[131I]iodobenzylguanine: improved synthesis and further evaluation of a potential agent for imaging of alkylguanine-DNA alkyltransferase

O(6)-Benzylguanine derivatives with suitable radionuclides attached to the benzyl ring are potentially useful in the noninvasive imaging of the DNA repair protein, alkylguanine-DNA alkyltransferase (AGT). Previously, O(6)-3-[(131)I]iodobenzylguanine ([(131)I]IBG) was prepared using a two-step approach; we now report its synthesis in a single step by the radioiododestannylation of O(6)-3-(trimethylstannyl)benzylguanine in 85-95% radiochemical yield. The in vitro specific uptake of [(131)I]IBG in DAOY human medulloblastoma cells, in TE-671 human rhabdomyosarcoma cells and a CHO cell line transfected to express AGT was linear (r(2) = 0.9-1.0) as a function of cell density. After intravenous injection of [(131)I]IBG in athymic mice bearing TE-671 xenografts, tumor uptake was 1.38 +/- 0.34% ID/g at 0.5 h and declined at 2 and 4 h. Preadministration of O(6)-(3-iodobenzyl)guanine (IBG) at 0.5 h increased uptake not only in tumor but also in several normal tissues. Notable exceptions were thyroid (p < 0.05), lung (p <0.05) and stomach. After intratumoral injection of [(131)I]IBG in the same xenograft model, the uptake in tumors that were depleted of AGT by BG treatment (165.8 +/- 27.5% ID/g) was about 60% of that in control mice (272.4 +/- 48.2% ID/g; p < 0.05).     

Erythrocyte phosphate composition and osmotic fragility in the Australian lungfish, Neoceratodus fosteri, and osteoglossid, Scleropages schneichardti

The packed cell volume (PCV), hemoglobin concentration (g/dl) and mean corpuscular volume (MCV) in the Australian lungfish, Neoceratodus fosteri, and in one of three Australian osteoglossids, Scleropages schneichardti, were 32.3 and 29.9; 10.5 and 10.0; and 407 and 176 micron 3 respectively. Total acid-soluble phosphates (TPi) from the red blood cells (RBC) of the lungfish and osteoglossid were 35.3 and 18.1 mumol/cm3 RBC respectively. Inorganic phosphate (Pi), adenosine triphosphate (ATP) and guanosine triphosphate (GTP) represented 16.4, 39.7 and 17.8% of the cell phosphates in the lungfish respectively. Inositol bisphosphate was not present in extracts of the red cells of N. fosteri, in contrast to the red cells of Lepidosiren paradoxa and Protopterus aethiopicus, in which it was first observed. In the osteoglossid, Pi and ATP represented 37.6 and 46.4% of the erythrocyte phosphate, respectively, with only traces of GTP present. ATP is the predominant organic phosphate in the red cells of both species. The osmotic fragility of erythrocytes of N. fosteri are quite resistant to hemolysis, with hemolysis beginning at 35-30 mM and a complete hemolysis occurring at 20 mM NaCl. The red cells of S. schneichardti begin to hemolyze at 95-90 mM with hemolysis continuing to completion at 60 mM NaCl.     

Effective bilayer expansion and erythrocyte shape change induced by monopalmitoyl phosphatidylcholine. Quantitative light microscopy and nuclear magnetic resonance spectroscopy measurements.

When human erythrocytes are treated with exogenous monopalmitoyl phosphatidylcholine (MPPC), the normal biconcave disk shape red blood cells (RBC) become spiculate echinocytes. The present study examines the quantitative aspect of the relationship between effective bilayer expansion and erythrocyte shape change by a newly developed method. This method is based on the combination of direct surface area measurement of micropipette and relative bilayer expansion measurement of 13C crosspolarization/magic angle spinning nuclear magnetic resonance (NMR). Assuming that 13C NMR chemical shift of fatty acyl chain can be used as an indicator of lateral packing of membrane bilayers, it is possible for us to estimate the surface area expansion of red cell membrane induced by MPPC from that induced by ethanol. Partitions of lipid molecules into cell membrane were determined by studies of shape change potency as a function of MPPC and red cell concentration. It is found that 8(+/- 0.5) x 10(6) molecules of MPPC per cell will effectively induce stage three echinocytes and yield 3.2(+/- 0.2)% expansion of outer monolayer surface area. Surface area of normal cells determined by direct measurements from fixed geometry of red cells aspirated by micropipette was 118.7 +/- 8.5 microns2. The effective cross-sectional area of MPPC molecules in the cell membrane therefore was determined to be 48(+/- 4) A2, which is in agreement with those determined by x-ray from model membranes and crystals of lysophospholipids. We concluded that surface area expansion of RBC can be explained by a simple consideration of cross-sectional area of added molecules and that erythrocyte shape changes correspond quantitatively to the incorporated lipid molecules.