24-hour study of thawed erythrocytes. Value of a protective solution

We have previously shown that thawed RBC concentrate can be stored at +4 degrees C during 9 days if resuspended in a synthetic medium: ESOC. We now report the in vitro evolution of thawed RBC stored with or without protective medium during the 24 hours legal time-limit. (Formula: see text) We show that without protection, the ATP and 2,3-DPG levels remain acceptable, but spontaneous or caused hemolysis is high. The level of free Hb is soon over the legal limit. The addition of our protective medium enhances ATP and hemolysis is strongly reduced. We conclude that a protective medium should be added to all thawed RBC concentrates.     

Deformability investigations of erythrocytes stored at different temperatures

The deformability changes of erythrocytes stored at different temperatures were characterized by a filtration procedure. The filterability index (FI) of erythrocytes cryopreserved at either -196 degrees C or -28 degrees C increased (deformability decreased) by about 9% and 40% respectively. For comparison with the cryopreserves the filterability of erythrocytes stored in liquid state at +4 degrees C were investigated. The filterability changes are discussed in comparison with the changes of MCHC during storage.     

Deformability characterization of erythrocytes stored in different resuspension media

The deformability changes of erythrocytes during 35 days of storage in different resuspension media were characterized by a filtration procedure. The filterability of erythrocytes stored in CDS-AG medium decreased drastically, whereas the filterability of erythrocytes stored in SAGM and SAGS medium decreased only slightly. The MCHC change of stored erythrocytes were discussed in relation to filterability effects.     

Deformability of stored erythrocytes as a criteria for their in vivo survival rate

The loss of deformability observed in erythrocytes stored as whole blood for 36 days (ACD-AG) or as buffy-coat free erythrocyte concentrate (EK) was characterized by measuring their filterability. During the first 3 weeks the index of filterability for ACD-AG erythrocytes increased only slightly and rose to about 140% of its initial value on the 36th day. In contrast, a heavy loss of deformability (increase of the filterability index to more than 600%) was detected for erythrocytes from EK, which, from a rheological point of view, is apt to raise doubts of using this stored blood. An incubation of 1 hour at 37 degrees C in fresh plasma did not result in improving the deformability. A cell volume loss of more than 20% connected with an increase of the inner viscosity to more than 400% was found to be the cause of this decrease of deformability. These rheological differences are also reflected in the 24 hours in vivo survival rate (SR), if the "early loss" of damaged erythrocytes immediately after transfusion is taken into account. Whereas the SR values of 80% for whole blood erythrocytes do not change significantly, the SR values for EK values can be found to reach 54% approximately.     

Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures.

High-frequency electric fields can be used to induce deformation of red blood cells. In the temperature domain T = 0 degrees to -15 degrees C (supercooled suspension) and for 25 degrees C this paper examines for human erythrocytes (discocytes, young cell population suspended in a low ionic strength solution with conductivity sigma(25 degrees) = 154 microS/cm) in a sinusoidal electric field (nu = 1 MHz, E0 = 0-18 kV/cm) the following properties and effects as a function of field strength and temperature: 1) viscoelastic response, 2) (shear) deformation (steady-state value obtained from the viscoelastic response time), 3) stability (by experimentally observed breakdown of cell polarization and hemolysis), 4) electrical membrane breakdown and field-induced hemolysis (theoretical calculations for ellipsoidal particles), and 5) mechanical hemolysis. The items 2-4 were also examined for the frequency nu = 100 kHz and for a nonionic solution of very low conductivity (sigma(25 degrees) = 10 microS/cm) to support our interpretations of the results for 1 MHz. Below 0 degrees C with decreasing temperature the viscoelastic response time tau(res)(T) for the cells to reach steady-state deformation values d(infinity,E) increases and the deformation d(infinity,E)(T) decreases strongly. Both effects are especially high for low field strengths. The longest response time of approximately 30 s was obtained for -15 degrees C and small deformations. For 1 MHz the cells can be highly elongated up to 2.3 times their initial diameter a0 for 25 degrees and 0 degrees C, 2.1a0 for -10 degrees C and still 1.95a0 for -15 degrees C. For T > or = 0 degrees C the deformation is limited by hemolysis of the cells, which sets in for E0(lysis)(25 degrees) approximately 8 kV/cm and E0(lysis)(0 degrees) approximately 14 kV/cm. These values are approximately three times higher than the corresponding calculated critical field strengths for electrically induced pore formation. Nevertheless, the observed depolarization and hemolysis of the cells is provoked by electrical membrane breakdown rather than by mechanical forces due to the high deformation. For the nonionic solution, where no electrical breakdown is expected in the whole range for E0, the cells can indeed be deformed to even higher values with a low hemolytic rate. Below 0 degrees C we observe no hemolysis at all, not even for the frequency 100 kHz, where the cells hemolyze at 25 degrees C for the much lower field strength E0(lysis) approximately 2.5 kV/cm. Obviously, pore formation and growth are weak for subzero temperatures.     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

Bending properties of wood treated with a new organic wood preservative system

This study investigated the effect of treatment of southern pine with a new generation, all organic wood preserving system on the mechanical properties in bending. Static bending tests showed no deleterious effects of treatment on property values when compared with untreated controls. Values for samples treated to 0.774 kg/m(3) total active ingredients (ai) were lower when compared to a lower retention (0.184 kg/m(3)) or to a standard copper azole system, but were not different than water-treated controls.     

Loss of preservative from a tuberculin solution in rubber stoppered vials fastened with different seals

A tuberculin purified protein derivative (PPD) solution containing 0.3% phenol as a preservative was dispensed in glass vials closed with rubber stoppers fastened in three different ways, namely with Tear-off seals, Flip-off seals and partial seals. After various times of storage at 5 degrees C and 37 degrees C, the phenol content in the tuberculin solution was determined. It was found that the Flip-off seals allowed the phenol to escape at a faster rate than the Tear-off seals and that vials closed with partial seals showed the highest loss of phenol. Although these losses were much more pronounced at 37 degrees C than at 5 degrees C, the phenol content at the latter temperature was, over a period of three years, within the limits of acceptability for tuberculin products capped with Tear-off or Flip-off seals. A loss of phenol also occurred from tuberculin solution stored at -28 degrees C in vials capped with either Tear-off or partial seals. In addition to the Tear-off and Flip-off seals other seals such as the "controlled score' Flip-off seal and the Alcoa Steri-Twist cap were evaluated for their imperviousness to air. Except for the Alcoa Steri-Twist cap none of the seals we have investigated were air tight and hence entirely satisfactory to prevent losses of phenol by evaporation from tuberculin products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic response ot introduction of intact and formalinized erythrocytes]

Experiments were conducted on mice; there was revealed an increase in the immunogenicity of erythrocytes following their immunization illustrated by the difference in the circulating antibody titres and the plaque count in the spleen of the animals, and also by the results of immunization with formalin-treated erythrocytes mixed with the cells of the peritoneal exudate. A conclusion was drawn on the expediency of the immunization with formalinized erythrocytes of rabbits--producers of the hemolytic serum.     

Hippocalcin: a new solution to an old puzzle

In hippocampal pyramidal neurons, calcium entry following an action potential burst results in a slow afterhyperpolarization (sAHP) that critically regulates subsequent excitability. Although this potassium current was described two decades ago, the mechanism whereby the rise in intracellular calcium generates the sAHP was, until now, not known. In this issue of Neuron, Tzingounis et al. now show that calcium binding to hippocalcin, a member of the NCS family, is one of the necessary steps involved in production of the sAHP.     

The hydration problem in solution biophysics: an introduction.

The background and purpose of the British Biophysical Society Discussion meeting, The Hydration Problem in Solution Biophysics, held at the University of Glasgow, 12 September 2000, is described, particularly in relation to previous meetings in this field. Whereas a study of the nature and dynamic properties of water associated with a molecule is an important topic by itself, the collection of papers based on this meeting focus mainly on its affect in interpreting biophysical data in terms of macromolecular shape in a solution environment, particularly under dilute and very dilute systems. The techniques considered are largely hydrodynamically or thermodynamically based and supplemented by molecular modeling strategies; but in the context of how these could be used in conjunction with techniques like X-ray crystallography, NMR and neutron scattering.     

Water is a preservative of microbes

Water is the cellular milieu, drives all biochemistry within Earth’s biosphere and facilitates microbe-mediated decay processes. Instead of reviewing these topics, the current article focuses on the activities of water as a preservative—its capacity to maintain the long-term integrity and viability of microbial cells—and identifies the mechanisms by which this occurs. Water provides for, and maintains, cellular structures; buffers against thermodynamic extremes, at various scales; can mitigate events that are traumatic to the cell membrane, such as desiccation–rehydration, freeze–thawing and thermal shock; prevents microbial dehydration that can otherwise exacerbate oxidative damage; mitigates against biocidal factors (in some circumstances reducing ultraviolet radiation and diluting solute stressors or toxic substances); and is effective at electrostatic screening so prevents damage to the cell by the intense electrostatic fields of some ions. In addition, the water retained in desiccated cells (historically referred to as ‘bound’ water) plays key roles in biomacromolecular structures and their interactions even for fully hydrated cells. Assuming that the components of the cell membrane are chemically stable or at least repairable, and the environment is fairly constant, water molecules can apparently maintain membrane geometries over very long periods provided these configurations represent thermodynamically stable states. The spores and vegetative cells of many microbes survive longer in the presence of vapour-phase water (at moderate-to-high relative humidities) than under more-arid conditions. There are several mechanisms by which large bodies of water, when cooled during subzero weather conditions remain in a liquid state thus preventing potentially dangerous (freeze–thaw) transitions for their microbiome. Microbial life can be preserved in pure water, freshwater systems, seawater, brines, ice/permafrost, sugar-rich aqueous milieux and vapour-phase water according to laboratory-based studies carried out over periods of years to decades and some natural environments that have yielded cells that are apparently thousands, or even (for hypersaline fluid inclusions of mineralized NaCl) hundreds of millions, of years old. The term preservative has often been restricted to those substances used to extend the shelf life of foods (e.g. sodium benzoate, nitrites and sulphites) or those used to conserve dead organisms, such as ethanol or formaldehyde. For living microorganisms however, the ultimate preservative may actually be water. Implications of this role are discussed with reference to the ecology of halophiles, human pathogens and other microbes; food science; biotechnology; biosignatures for life and other aspects of astrobiology; and the large-scale release/reactivation of preserved microbes caused by global climate change.     

Developing oscillatory flow in a circular pipe: a new solution

The problem of oscillatory flow in a circular pipe was analyzed by Atabek and associates more than two decades ago. Their formulas for velocity and pressure distributions in developing pipe flows under oscillatory conditions have been often cited. However, the application of these formulas for flow field computations requires a rather complex procedure involving plotting of a set of curves and predetermination of the phase angles. This paper presents a method using the imaginary argument of the Bessel function to solve the Navier-Stokes equations. A different set of solution formulas are obtained. A comparison of the formulas obtained in this paper with those of Atabek shows that the former is considerably simpler and more convenient to use in flow computations. Numerical results computed using this paper's formulas are consistent with Atabek's and with the experimental measurements.     

Metabolic control: a new solution to an old problem

The phenomenon whereby the presence of oxygen regulates the rate of glucose metabolism was first described by Louis Pasteur. A novel mechanism has now been discovered, involving the AMP-activated protein kinase cascade, that can account for the Pasteur effect in ischaemic heart muscle.     

Honey as a natural preservative of milk

The anti-bacterial property and preservative nature of honey has been studied by evaluating the role of hydrogen peroxide in these properties, against bacterial strains isolated and identified from pasteurized milk samples. The antibacterial property of honey examined by agar incorporation assay and turbidometry, indicated a concentration dependent inhibition of bacterial growth in all catalase negative strains in comparison with catalase positive strains, highlighting a probable role of hydrogen peroxide. Samples of commercial milk stored at 40C in presence of honey were shown to inhibit opportunistic bacterial growth better compared to samples stored without honey. Due to the bactericidal property of hydrogen peroxide and its preservative nature, honey which is chiefly a combination of various sugars and hydrogen peroxide, can be used a preservative of milk samples.     

Chromatographic identification of a new subform of carbonic anhydrase in rabbit erythrocytes

In addition to the two forms of carbonic anhydrase (CA) known to occur in rabbit erythrocytes, a third enzyme species has been isolated and partially characterized. The three Chromatographic forms elute from diethylaminoethyl-Sephadex roughly in the proportions of 3:5:2, in that order. They do not appear to be interconvertible. Their molecular weights are approximately 30,000. Initial kinetic studies of ester hydrolysis and of CO₂ hydration by these carbonic anhydrases identify the first peak to elute as a low-activity form (CA I) and the following two as high-activity forms (CA II, CA II′). Inhibition studies indicate that equal concentrations of all three enzyme species are equally inhibited by equal concentrations of benzenesulfonamide or acetazolamide. The amino acid compositions of the three species support their identification as high- and low-activity forms and indicate substantial differences between CA I and CA II or CA II′, while the latter two differ only slightly. Peptide maps obtained from tryptic digests show considerable differences between the peptides of the high- and low-activity forms, while the peptides of CA II and CA II′ are almost indistinguishable. The available evidence suggests that the newly isolated CA II′ species is a subform of the major high-activity isoenzyme and that a very slight difference in amino acid composition is responsible for a net charge difference between CA II and CA II′, which is reflected in their different Chromatographic properties.