Enhanced reduction of nitrous oxide by Pseudomonas denitrificans with perfluorocarbons

Nitrous oxide reduction and nitrogen production by Pseudomonas denitrificans, as well as culture growth rates all increased 2-3 fold when cultured in the presence of perfluorocarbon emulsions (10% v/v) as compared to control cultures grown in the absence of perfluorocarbons. Initial nitrous oxide concentrations for consecutive experiments were 0.7 and 1.2 mM respectively.     

Supercooling and nucleation of ice in single cells

An emulsion droplet formation procedure was employed to isolate yeast cells and, in separate experiments, human red blood cells, one from another in individual droplets, and to segregate extraneous materials catalyzing the formation of ice. Emulsification succeeded in isolating the cells and permitted the observation of the supercooling of droplets containing cells whereby each droplet was observed to nucleate ice at a temperature that depended only upon the components of the droplet. The droplet formation procedures were characterized. It was shown that the surface coatings and the carrier fluids used in the preparation of the emulsions did not act as ice nucleators. It was, in this manner, possible to study the nucleation of ice brought about by supercooling and homogeneous nucleation in the volume of the droplet or by the catalysis of nucleation on or in the cells contained in the droplets. It was shown that yeast cells and red blood cells could each be supercooled to about ?40 °C in short-term experiments. The results also revealed that yeast cells did not store for infinite times at temperatures above the observed upper limit of homogeneous nucleation. The yeast cells died at rates that were exponential functions of time at ?20, ?22.5, ?25, ?29 and ?33 °C. The temperature dependence of the death rate did not correspond to a process with a normal Arrhenius activation energy. The temperature dependence did, however, suggest a potentiated heterogeneous catalysis of ice resulting in the death of the yeast cells.     

Dielectric Properties of Water-liquid Paraffin Emulsion Systems at the Microwave Frequency of 9.4 GHz

The state of water contained in emulsions, particularly in o/w emulsions, was studied as a model of water orientation at the peripheries of biomembranes. Dielectric measurements made at microwave frequency on emulsions containing water and liquid paraffin in various ratios with emulsifiers revealed that the o/w emulsions possessed considerably reduced dielectric loss as compared with theoretical values obtained in accordance with the Maxwell-Wagner model, while the dielectric properties of w/o emulsion were in good agreement with the theoretically expected values. The observations seem to be explained by assuming changes in the state of water in the oil-water interfacial layer in o/w emulsions. The preparation of stable emulsions for use in this study is also discussed.     

Solid-phase extraction of phospholipids from hemoglobin solutions using Empore styrene–divinylbenzene disks

Styrene–divinylbenzene Empore disks were investigated for the extraction of phospholipids from red blood cells or aqueous solutions of hemoglobin as a means to reduce the time and solvent use required in sample preparation. Red blood cells are the source for hemoglobin used in the preparation of a hemoglobin-based oxygen carrier which is being developed to replace blood in transfusion therapy. Phospholipids are a major component of the membrane of red blood cells, and are toxic when administered directly into the vasculature. Sensitive analytical methods are required to detect phospholipids to ensure that concentrations in purified hemoglobin are well below toxic levels. This requires isolation from large volumes of purified hemoglobin solutions. The method described utilizes Empore disks to extract phospholipids from 30 ml of stroma free Hb preparations. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and sphingomyelin were recovered with an average of 92% yield. The recovery of phosphatidylserine was 65%. The use of solvent and time required for sample preparation were reduced by an average of 80% relative to liquid–liquid extraction. The capacity of the 47-mm disk for the total of five phospholipids exceeds 0.3 mg. The method has been used for quantitation of phospholipids in red blood cells and stroma free hemoglobin solutions.     

Blood as a near-"ideal" emulsion: a retrospective on the concept of the red cell as a fluid drop, its implications for the structure of the red cell membrane

Although the question whether the red cell is fluid or solid has been discussed since 17th century, it was the author's measurement of the relative viscosity of blood in 1960's that supplied the first direct evidence that the red cell interior is fluid. Furthermore, through his application of the equations of Taylor and, later, Oldroyd, to this problem, it became evident that, for the red cell to exhibit fluid-drop-like behavior, the membrane must also be fluid. This led to his concept of the red cell membrane as a complex two-phase structure (lipoprotein micelles and two-dimensional protein networks) which was similar to the one accepted nearly a decade later. The requirements of the theory of ideal emulsions that the shear stress be transmitted into the cell interior via low viscosity membrane, are met in the later work of other investigators using the concept of a tank-treading membrane having viscoelastic properties. This paper reviews the original work of the author which led to the development of an equation for the relative viscosity of blood as a function of volume concentration, C: nr = (1 - TkC)-2.5, valid at shear rates above 180 sec-1, in which T is the Taylor factor which gives a measure of fluidity of the red cell, and k is a plasma trapping factor. Both T and k increase with increasing rigidity of the red cell. Finally, the effect of the membrane viewed as a complex two-phase fluid, on the rheology of the red cell is discussed.     

Leukocyte-depleted blood: a comparison of available preparations.

Febrile nonhemolytic transfusion reactions due to leukoagglutinins are frequently seen in patients who have been given multiple blood transfusions. To prevent or reduce the severity of these reactions, leukocyte-poor blood (that containing fewer than 0.3 X 10(9) leukocytes per unit) is frequently requested by clinicians. Four methods commonly used in Canada to produce leukocyte-poor blood were examined for their relative effectiveness and appropriate use. The mean total leukocyte count per unit was reduced to 0.22 X 10(9) in buffy-coat-poor red blood cell preparations produced by centrifugation with the blood bag inverted, to 0.19 X 10(9) by perfusion through an Imugard filter, to 0.21 X 10(9) by the use of an IBM 2991 automated cell washer and to 0.13 X 10(9) with the use of frozen blood. The proportion of red cells recovered varied from 62% with the inverted-spin method to 85% with the use of frozen blood. Comparison of these data and the percentage of leukocytes removed, the shelf life of the product, the cost of supplies and the preparation time indicated that the use of sophisticated machinery, such as the IBM cell washer, or of glycerolization plus washing of frozen cells is not warranted for most patients. Instead, patients who have febrile nonhemolytic transfusion reactions should initially be treated with a leukocyte-poor red cell preparation produced by the inverted-spin method; only if such reactions recur should the blood bank be requested to provide filtered, washed or frozen red cells.     

脱磷脂牛血红蛋白的制备

研究了一种新的脱磷脂血红蛋白制备方法。在2%、5%PEG4000或2%、5%PEG10000作共溶剂的情况下,利用疏水相互作用色谱基本上完全除去了新鲜牛血红细胞裂解液中的磷脂类成分,其中以5%PEG4000为共溶剂时,血红蛋白的回收率最高,达85.0%,在血红蛋白存在下所用的苯基琼脂糖-6B型疏水介质的吸附容量为86.6 mg磷脂/mL介质。经过疏水作用色谱后,血红蛋白的P50是33864Pa,Hill系数是2.54,较好地保存了血红蛋白的生物活性。对疏水作用色谱的脱磷脂机理以及PEG的保护机制进行了讨论。  血红蛋白,疏水作用色谱,PEG,磷脂,共溶剂     

A new method to study shape recovery of red blood cells using multiple optical trapping.

In this new method for studying the shape recovery of deformed red blood cells, three optical traps ("optical tweezers") induce a parachute-shaped red cell deformation, which is comparable to the deformation in small capillaries. The shape recovery is recorded, and a relaxation time is obtained for each individual red blood cell. The sensitivity of this technique for the detection of differences in relaxation times is demonstrated on subpopulations of density-separated red blood cells: "young" cells have shorter (162 ms) and "old" cells have longer (353 ms) relaxation times compared with the total population (271 ms). The relaxation time is remarkably shorter (114 ms) when the plasma surrounding the cells is replaced by a phosphate-buffered saline solution. The main advantages of this technique are the relatively short measuring and preparation time and the physiological type of deformation and shape recovery in which all relevant cell properties play a role. Therefore, especially when automated further, the technique may be a powerful tool for the study of (sub)populations of pathological red blood cells.     

Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets,erythrocytes, and leukocytes

Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.     

Stimulation of calcium-sodium exchange in dog red blood cells by hemolysis and resealing

Osmotic hemolysis and resealing greatly increase calcium influx in dog red blood cells. The resealed ghosts show a saturable calcium entry pathway with complex kinetics. As expected for a calcium-sodium exchanger, calcium uptake is stimulated by internal sodium and inhibited by external sodium. Compared to fresh, intact red cells the resealed ghost calcium-sodium exchanger is less responsive to quinidine and to alterations in medium tonicity. The differences in calcium uptake rate among cells from different donors are minimized in the ghost preparation. There are several ways to stimulate sodium-dependent calcium movements in these cells, of which hemolysis-resealing is the most potent. The results of these and previous studies suggest that dog red blood cells have a latent capacity for calcium-sodium exchange.     

Interaction of bacteria and red blood cells

The interaction of staphylococci, streptococci, meningococci, enterobacteria, leptospires and other microorganisms with red blood cells is considered. Three forms of the interaction of bacteria and red blood cells are discussed: adhesion, the influence of secretory factors on red blood cells, the action of pathogenic bacteria on hemoglobin. The applied aspects of the interaction of bacteria and red blood cells in the human body are presented in accordance with the results of clinical and laboratory studies.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

Reduced cytotoxicity of polyhexamethylene biguanide hydrochloride (PHMB) by egg phosphatidylcholine while maintaining antimicrobial efficacy

Liposomes or oil-in-water emulsions containing egg yolk phosphatidylcholine (EPC) were combined with aqueous polyhexamethylene biguanide hydrochloride (PHMB). The bactericidal activity of these preparations against Pseudomonas aeruginosa and Staphylococcus aureus as well as their cytotoxicity on cultured murine fibroblasts (L929 cells) was then assayed for either 30 min or 60 min in the presence of cell culture medium containing 10% fetal bovine serum as surrogate for wound fluid. We used two assay designs: in the first bactericidal activity and cytotoxicity were determined in separate experiments; in the second both were determined in one experiment. Combining PHMB and EPC containing o/w emulsions or liposomes protects mammalian cells without neutralizing the antiseptic effect. From all tested combinations the o/w emulsions containing 0.05% PHMB proved to be superior in this respect to the aqueous preparation.     

Use of perfluorocarbon emulsions in cell culture.

Perfluorocarbon emulsions were applied to hybridoma cultures grown in tissue culture tubes and column bioreactors. The oxygen transfer enhancement effect of perfluorocarbon emulsions was clearly demonstrated by the higher cell densities obtained in emulsion-supplemented systems. In addition, perfluorocarbon emulsions were shown to provide better cell suspension in a low-shear environment. The study in column bioreactors also suggested a cell protective effect of the employed perfluorocarbon emulsions in reducing the damage to cells by gas bubbles.     

Parallel microchannel-based measurements of individual erythrocyte areas and volumes

We describe a microchannel device which utilizes a novel approach to obtain area and volume measurements on many individual red blood cells. Red cells are aspirated into the microchannels much as a single red blood cell is aspirated into a micropipette. Inasmuch as there are thousands of identical microchannels with defined geometry, data for many individual red cells can be rapidly acquired, and the fundamental heterogeneity of cell membrane biophysics can be analyzed. Fluorescent labels can be used to quantify red cell surface and cytosolic features of interest simultaneously with the measurement of area and volume for a given cell. Experiments that demonstrate and evaluate the microchannel measuring capabilities are presented and potential improvements and extensions are discussed.     

High Susceptibility of Strain A Mice to Endotoxin and Endotoxin-Red Blood Cell Mixtures.

Heppner, Gloria (University of California, Berkeley), and David W. Weiss. High susceptibility of strain A mice to endotoxin and endotoxin-red blood cell mixtures. J. Bacteriol. 90:696-703. 1965.-Strain A mice were shown to be considerably more susceptible to lethal effects of endotoxin lipopolysaccharide (LPS) than mice of several other strains. Complexes of sublethal quantities of LPS and sheep red blood cells were synergistically toxic for strain A mice. Separate administration of sheep red blood cells and heat-killed salmonellae, in either order and as long as 24 hr apart, also proved to be synergistically lethal for strain A mice, but not for R(III) animals studied comparatively. Sheep red blood cell lysates possessed the ability of the intact cells in forming lethal combinations for strain A mice with killed salmonellae. Strain A red blood cell-killed salmonellae complexes were also lethal for strain A mice, but less so then complexes made with sheep red blood cells. A x R(III) F(1) hybrid animals showed the same resistance characteristics as the resistant R(III) parental strain. Possible explanations for these findings are suggested, and their relevance to an immunological mode of action of endotoxin lethality is discussed.     

Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.     

Nanobiotechnological modification of hemoglobin and enzymes from this laboratory

Polyhemoglobin is formed by the nanobiotechnological assembling of hemoglobin molecules into soluble nanodimension complex. A further step involves the nanobiotechnological assembly of hemoglobin, catalase and superoxide dismutase into a soluble nanodimension complex. This acts both as oxygen carrier and antioxidant to prevent the oxidative effects of hemoglobin. A further step is the preparation of nanodimension artificial red blood cells that contain hemoglobin and all the enzymes present in red blood cells. Other approaches include a polyhemoglobin-fibrinogen that acts as an oxygen carrier with platelet-like activity, and a polyhemoglobin-tyrosinase to retard the growth of a fatal skin cancer, melanoma.     

Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.