Thinking about strategies during, before, and after making a decision

Oxidative radicals are demonstrably produced in malaria-infected erythrocytes. In order to verify the biochemical origin of these radicals, erythrocyte lysate was brought to acid pH to mimic the environment of the parasite food vacuole into which host cell cytosol is transferred during parasite feeding. Oxyhemoglobin, but not deoxyhemoglobin, is rapidly converted to methemoglobin at rates which decline with increasing pH. The rate of conversion is further increased in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and the extent of inhibition of the lysate catalase increases upon acidification, implying that H2O2 is thus produced by the spontaneous dismutation of superoxide radicals generated during methemoglobin formation. Intact Plasmodium falciparum trophozoite-infected human red blood cells (TRBC) were shown to produce H2O2 and OH radicals about twice as much as normal erythrocytes, as evidenced by the inhibition of endogenous catalase activity in the presence of 3-AT and the degradation of deoxyribose, respectively. Increased H2O2 levels and catalase activity were found in both host cell and parasite compartments. No increase in H2O2 production over that observed in uninfected erythrocytes could be detected at the ring stage when host cell digestion is absent. H2O2 and OH radicals production in TRBC was considerably reduced when digestion of host cell cytosol was inhibited either by antiproteases (which reduce the proteolysis of imported catalase) or by its alkalinization with NH4Cl (which reduce methemoglobin formation). These results suggest that reactive oxygen species are produced in the parasite's food vacuole during the digestion of host cell cytosol, and are able to egress from the parasite to the host cell compartment.     

Human lymphoblastoid cell line producing specific antibody against Rh-antigen D

A lymphoblastoid cell line producing specific anti-Rh antibody against D determinant was established by Epstein-Barr virus (EBV) infection. The lymphocytes were from an immunized male donor with high titre of anti-D antibody. The cells were preselected by rosetting them with Rh-positive erythrocytes before EBV infection. The cell line produced specific antibody of IgG class, namely IgG1 subclass, but it also produced nonspecific IgM and IgG. By rerosetting the cell line, the specific antibody-producing population could be enriched and the antibody titre increased in the culture supernatant to the level of immune sera.     

Influence of immunoadjuvants and a promiscous T-cell determinant on the immunogenicity of RESA peptide antigen of P. falciparum

Synthetic peptide antigens representing the repeat sequences of malarial antigens showed poor immunogenicity and protection in clinical trials. In the present study, RESA, an asexual blood stage antigen, containing (EENVEHDA)2 and (DDEHVEEPTVA)2 sequences were chemically linked to a promiscous T-cell determinant (CS.T3) of the circumsporozoite protein of P. falciparum. The synthetic constructs either alone or coentrapped with immunoadjuvants (nor muramyl dipeptide/lauroyl tetrapeptide) were administered in liposomes to mice of varying genetic background and the immunogenicity of different formulations were compared under identical experimental conditions. The RESA peptide formulations containing the T-cell determinant and the adjuvants generated high titre and affinity antibodies in all the strains, as compared to peptide(s) alone. The booster immunization induced a strong anamnestic response in each group. Though the major IgG isotype is of IgG1 and IgG2b interestingly, formulations containing CS.T3 have a higher proportion of cytophilic IgG2b isotype. There was a significant fall in the levels of IgG2b isotype while IgG1 levels were maintained same in the third bleed (day 60, without booster immunization). The mixed peptide group preparation containing the adjuvant is found to be a better immunogen than that of respective peptides itself. The in vitro merozoite reinvasion inhibition assay showed 76-96% inhibition with the formulations containing RESA peptide(s)-CS.T3 and the adjuvant, while with peptides alone the inhibition was 50-56%. This study highlights the importance of an alternative approach for developing peptide based immunogen against malaria.     

Histamine metabolism of the guinea-pig gastric mucosa

(1) Preparative dodecyl sulfate gel electrophoresis of human erythrocyte membrane proteins has been used to isolate dodecylsulfate band 3 containing the M,N-glycoprotein and the major "intrinsic" membrane protein (Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617; Bretscher, M.S. (1971) J. Mol. Biol. 59,351-357; Bretscher, M.S. (1971) Nat. New Biol. 231, 229-232 and Marchesi, V.T. and Andrews, E.P. (1972) Science 174, 1247-1248). Subsequent isoelectric focusing in polyacrylamide gels containing Triton X-100 separates these two entities and allows their simultaneous purification. (2) The proteins thus obtained retain their antigenic properties. They are pure according to electrophoretic and immunoelectrophoretic criteria. However, crossed immunoelectrophoresis yields evidence for molecular microheterogeneity of the major "intrinsic" protein. (3) Analyses utilizing crossed immunoelectrophoresis with antibodies absorbed with intact erythrocytes show that the major "intrinsic" protein possesses antigenic determinants on both membrane surfaces and therefore spans the erythrocyte membrane. All determinants of the M,N-glycoprotein detectable with our antibodies were found solely on the exterior membrane surface. (4) Neither the major "intrinsic" membrane protein nor the major M,N-glycoprotein bound significantly to concanavalin A in crossed immunoaffinoelectrophoresis.     

Identification of a C4 Subcomponent on C3d-coated erythrocytes

Test erythrocytes (E) used to evaluate anti-complement (C') antiglobulin sera have not been adequately standardized. This report describes a previously unrecognized C4-derived antigen (temporarily called X-Ag) found on E generally believed to be coated only with the C3d subcomponent of C3, X-Ag occurred on all E coated in vitro with C' by low ionic strength-sucrose or cold agglutinin methods and on E from ten of ten patients whose cells had been C' coated in vivo. It was not removed by incubating these cells with trypsin or fresh compatible serum. This antigen was found on "C4-only-coated" red blood cells made with normal or congenitally C2-deficient serum but not on cells similarly prepared with congenitally C4-deficient serum. It was not identified on E coated with C' via the alternate pathway, normal trypsinized cells, nor cells coated only with IgG. Absorption experiments utilizing purified complement components and subcomponents and G200 Sephadex fractions of normal human serum strongly suggest that X-Ag is a subcomponent of C4(C4d). These results show that at least one C' subcomponent other than C3d occures on both in vitro and in vivo C3d-coated erythrocytes and must be taken into account when such cells are used to evaluate antiglobulin reagents.     

Processes involved in the computation of a shape description.

The encoding of shape information from spatially separate items was investigated in 3 experiments. In Experiment 1a, participants had to search for an inward-pointing arc among a set of outward-pointing arcs; participants in Experiment 1b were given the opposite task. Search was efficient if distractor arcs pointed away from fixation and there was curvilinearity between neighboring end-terminators (Experiment 1a) but relatively inefficient if distractor arcs pointed toward fixation (Experiment 1b). In Experiments 2 and 3, connecting end terminators did not change performance. Results reflect the involvement of mechanisms of shape encoding. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Dynamic CT measurement of cerebral blood flow: a validation study

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [125I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28-40%, (ii) the RGD peptide or MoAbs against integrin alpha v beta 3 or the calcium binding region of TSP inhibited binding by 18-28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.     

Inhibition of erythrocyte selenium-glutathione peroxidase by auranofin analogues and metabolites

The effect of gold ligation on the inhibition of bovine erythrocyte selenium-glutathione peroxidase (GSH-Px) was examined. The anti-arthritic drug auranofin [2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S)(triethylp hosphine) gold(I)] (Et3PAuSATg) and its analogue, Et3PAuCl, exhibited experimentally equivalent Ki values (11.6+/-0.8 and 10.8+/-0.5 microM, respectively), despite the greatly disparate affinities of their ligands for gold(I): 2,3,4,6-tetra-O-acetyl-1-thiolato-beta-D-glucopyranose (ATgS-) > Cl-. This similarity reflects ligand exchange reactions that generate the glutathione complex Et3PAuSG from the excess glutathione (GSH, 1 mM) used in the assay. The Ki values for bis(glutathionato)gold(l) (Au(SG)2-) and gold(I) thioglucose (AuSTg) were also found to be equal (2.8+/-0.4 and 2.4+/-0.5 microM, respectively). This confirms the previous postulate of Chaudiere and Tappel (J Inorg Biochem 20: 313-325, 1984) that Au(SG)2- is generated from AuSTg in the presence of excess glutathione. Since auranofin metabolites accumulate in red blood cells, the inhibition of intracellular GSH-Px was examined by using intact erythrocytes. There was greater inhibition of the reaction when the cells were resuspended in isotonic buffer than in whole blood, because serum albumin in the latter competes for the auranofin and decreases the uptake by erythrocytes. After correction for the extent of gold uptake, the Ki values were determined to be the same as those observed for Au(SG)2- in the extracellular assay, indicating loss of both the Et3P and ATgS- ligands from auranofin. Thus, the inhibition of GSH-Px by gold complexes is dependent on their ligation, and the ultimate gold(I) compound that interacts with erythrocyte GSH-Px in intact red cells, Au(SG)2-, is radically different from the original auranofin molecule.     

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.     

Enrichment of immunoglobulin binding Plasmodium falciparum-infected erythrocytes using anti-immunoglobulin-coated magnetic beads

It has been shown that nonimmune, human immunoglobulins are bound to the surface of certain strains of Plasmodium falciparum-infected erythrocytes. We describe a novel way of enriching parasitized red blood cells (pRBC) for immunoglobulin binding/rosette formation using Dynabeads coated with antibodies raised against human immunoglobulins. Whole P. falciparum cultures were mixed with the precoated beads for approximately 120 min at room temperature, and the bound pRBC were isolated by magnetic force. The nonbound cell fraction contained ring-infected pRBC, immunoglobulin-negative, trophozoite-infected pRBC, and uninfected erythrocytes. A consistent elevation in the immunofluorescence and rosette formation rates of 100% and 86% respectively, was detected after the first enrichment and subcultivation. Protein A or G were also found to support binding of pRBC through surface-expressed immunoglobulin. The Dynabead technique is a novel way of enriching pRBC based on the immunoglobulin-binding capacity of the infected erythrocyte.     

Visual misalignment in arc and chevron figures.

Five experiments investigated an apparent misalignment effect in 90° arc figures. Preliminary observations showed that the effect occurs also in chevron figures, in an afterimage of the arc figure, and haptically in arc- and chevron-shaped objects. The experiments showed that the effect is greater with 3 radial lines than with 2, absent without them, and present in a figure consisting of only 3 radial lines. The effect with arc figures was consistently greater than that with chevron figures, a difference found not to be due to an apex marking the midpoint of the latter, and it was of intermediate size in figures with 1 arc boundary and 1 chevron boundary. The misalignment was also greater in narrow, elongated figures. The issues singled out for discussion are the effect of context on the misalignment effect with 3 radial lines, a possible explanation in terms of perceptual compromise, the difference in the effect between arcs and chevrons, and the relationship between this illusion and the Morinaga illusion. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Dynamic adhesion of erythrocytes to glass. Comparative study of the influence of serum in low concentrations

Adhesion of 51Cr-labeled rat erythrocytes was examined in a layer of glass beads in a dynamic system. The kinetics of erythrocyte retention in the bead layer was found to change with time. Adhesion decreased with time at all the studied rates of suspension flow through the layer (within the range 1-5--0-4 cm/min), both in presence and absence of serum in the medium. Serum in low concentrations of 20--200 microgram of protein per ml considerably inhibited dynamic adhesion of erythrocytes to glass. The higher the serum concentration in a medium (at a definite flow velocity), the quicker the decrease of adhesion with time, and the stronger the dependence of the kinetics of erythrocyte retention on the flow velocity of the cells. Mean adhesion of erythrocytes after 17-min perfusion of the layer (at 200 microgram of serum protein per ml of medium) seems to be independent of the concentration of the cells in the suspension flowing into the layer within the range of studied concentrations, 2 x 10(6)--20 x 10(6) cells/ml. However, mean adhesion of erythrocytes deprived of serum seems to be dependent on the cell concentration.     

Lipid peroxidation and antioxidant status in the liver, erythrocytes, and serum of rats after methanol intoxication

Lipid peroxidation products measured as a malondialdehyde and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), and concentrations of ascorbic acid, alpha-tocopherol, and glutathione (GSH) were measured in the liver, erythrocytes, and serum of rats 6, 14, and 24 h and 2, 5, and 7 d after treatment with 3 g methanol/kg. GSH-Px and GSSG-R activities, GSH level, and ascorbate concentration in the liver, erythrocytes, and blood serum were significantly decreased. In addition, SOD and alpha-tocopherol in erythrocytes were diminished, while malondialdehyde (MDA) in liver, erythrocytes, and serum were elevated. Further, erythrocyte counts, hemoglobin levels, hematocrit, and mean corpuscular volume (MCV) were reduced. These results indicate that methanol intoxication in rats leads to an increase in the lipid peroxidation and impairment in the antioxidant mechanisms in liver, erythrocytes, and blood serum.     

Erythrocyte dehydration in pathophysiology and treatment of sickle cell disease

A prominent feature of sickle cell disease is the presence of cells with markedly increased sickle cell hemoglobin concentration, as a consequence of the loss of potassium, chloride, and water from the erythrocyte. Because of the extreme dependency of the kinetic of polymerization on sickle cell hemoglobin concentration, these dehydrated erythrocytes have an increased tendency to polymerize and sickle. Thus blockade of the loss of potassium from the erythrocyte should prevent the increase in sickle cell hemoglobin concentration and reduce sickling. The availability of this potential therapeutic option is based on a detailed knowledge of the mechanisms leading to cell dehydration. Two ion transport pathways, the K-Cl cotransport and the Ca(2+)-activated K+ channel, play a prominent role in the dehydration of sickle erythrocytes. Possible therapeutic strategies include inhibition of K-Cl cotransport by increasing erythrocyte Mg2+ content and inhibition of the Ca(2+)-activated K channel by oral administration of clotrimazole.     

Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems

Erythrocyte aggregation, which plays an important role in the physiological behavior of blood fluidity, was found to be enhanced in hypertension and hypercholesterolemia. While the role of macromolecule bridging force has been widely described, cellular factors related to membrane sialic acid content, which might contribute to the negative charge of cell surface causing the repulsion of erythrocytes, have been less studied. Cell age-dependent changes in membrane sialic acid content (in micromoles per gram of integral membrane protein) were investigated in 24 normotensive and 24 hypertensive matched subjects, each divided into 2 identical subgroups according to a cutoff of 6.2 mmol/L serum cholesterol. A progressive and significant (P<0.001) decrease in membrane sialic acid content associated with an increase (P<0.001) of disaggregation shear rate threshold (laser reflectometry in the presence of dextran) were observed with increased erythrocyte density (erythrocytes fractionated by density using ultracentrifugation) in both normotensive and hypertensive groups regardless of the cholesterol level. However, disaggregation shear rate threshold was significantly higher and sialic acid content was lower (P<0.001) in both hypertensive and normotensive subjects with hypercholesterolemia compared with either normotensive or hypertensive subjects with low cholesterol, respectively. A high membrane sialic acid content variance, beginning in the younger erythrocytes, was due mainly to triglyceride and LDL cholesterol levels (R2=0.49 for low, R2=0.43 for middle, and R2=0.54 for high densities, ie, young, mean, and senescent erythrocytes, respectively). We conclude that an early decrease in erythrocyte sialic acid content may influence the rheological properties of blood by increasing the adhesive energy of erythrocyte aggregates.     

Functional analysis of the guanylate kinase-like domain in the synapse-associated protein SAP97

The effect of cations on the kinetics of hemolysis caused by organotin compounds was studied. The ions used in the investigation diminish or totally inhibit hemolysis of red cells induced by organotin compounds. The degree of inhibition depends both on the kind of ion and the compounds that induce hemolysis. The ions Zn2+, Co2+, and Cd2+ present in the medium at 50 microM concentration totally protect the erythrocytes against hemolysis induced by the compound (C3H7)3SnCl. The study has also shown the monovalent ions K+ and trimethyldodecylammonium bromide are less potent inhibitors of hemolysis than divalent ions, which is not the case for two non-ionic organotin compounds only. The studies performed indicate that hemolysis induced by organotin compounds is inhibited due to electrostatic interaction between the cations selected and erythrocyte membrane.     

Fractionation of human erythrocyte membranes. Presence of the nucleoside transport complex in an insoluble residue

(1) Human erythrocyte membranes, when dialysed against water at pH 9.5, were partly solubilized, losing 80% of the membrane proteins and 65% of the membrane lipids. Sodium dodecyl sulphate gel electrophoresis of the particulate material revealed selective removal of proteins from the membrane. (2) The lipid-rich particulate material remaining retained the ability to bind specifically the nucleoside transport inhibitor, nitrobenzylthioinosine, previously shown to bind selectively to the nucleoside transport mechanism of whole erythrocytes and erythrocyte ghosts.     

Analysis of the short tandem repeat systems HumVWA and HumF13B in a population sample from northern Thailand

Adenylate kinase activity originating from erythrocytes has been shown to be distinct from muscle adenylate kinase or myokinase activity, until now considered to be identical enzyme activities. The two activities can be differentiated by electrophoretic fractionation, thus making it possible to quantify the erythrocyte adenylate kinase activity present in serum.     

A computer-based statistical pattern recognition for Doppler spectral waveforms of intracranial blood flow

The effects of 2 months treatment with simvastatin (40 mg, 20 mg p.o. daily) or placebo on erythrocyte membrane cholesterol content and acyl chain composition have been studied in 36 patients with a clinical history of atherosclerosis enrolled in the Oxford Cholesterol Study. All patients received advice corresponding to a standard phase 1 cholesterol-lowering diet. As expected the mean serum total cholesterol fell substantially (-26.5%, 20 mg simvastatin, P < 0.05; -32.7%, 40 mg simvastatin, P < 0.05) compared to placebo (-6.3%, ns). However, mean erythrocyte cholesterol content did not change significantly in any group (2 months therapy: 20 mg simvastatin, -0.62%; 40 mg simvastatin, +2.2%; placebo, -4.2%). Erythrocyte cholesterol was also unaltered after 5 months of therapy. Erythrocyte osmotic fragility was unchanged in the treatment and placebo groups. In the placebo group dietary advice alone was associated with a significant increase in the linoleic acid content of erythrocytes from 9.4 mole% of total acyl chains to 11.8 mole% (P < 0.05). Treatment with simvastatin was associated with an increase in the arachidonic acid content of the erythrocyte membrane from 12.2 to 15.3 mole% (P < 0.05). Treatment with simvastatin does not alter erythrocyte cholesterol content, but does alter acyl chain distribution. These results suggest that the chemical potential of cholesterol in serum is not markedly altered by HMG-CoA reductase inhibition.