Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated at 37 degrees C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34 +/- 2.2 nmol g-1 ghosts with a dissociation equilibrium constant (37 degrees C) Kdm 1.38 +/- 0.15 fold Kd of albumin binding Kdm is temperature independent and approximately 7-8 nM. Exchange efflux kinetics at 0 degrees C to buffers of various albumin concentrations ([BSAy]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450 +/- 0.018 and the rate constant of unidirectional flux from inside to outside is 0.067 +/- 0.01 s-1. The rate constant of flux from the extracellular side of the membrane to BSAy increases with the square root of [BSAy] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL-BSA complex at 0 degrees C of 0.0063 +/- 0.0003 s-1. Exchange efflux from ghosts containing four different [BSAi] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rate coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.     

Plasma and red cell lipids in sickle cell disease

Lipids, in particular phospholipids, are essential components of membrane systems, and the measurement of phospholipids and cholesterol in plasma and tissues is helpful in diagnosis. Phospholipids represent about 60 to 70% of total red cell (RBC) lipids, while about 25% is free cholesterol. Lipids in RBC are present in a dynamic state of equilibrium, and the RBC have the capacity for rapid exchange of lipids with plasma in several ways. The present study examined the cholesterol and phospholipid levels of plasma and erythrocytes in male patients with sickle cell anemia and in healthy male individuals of comparable age. This was performed with a view to detecting possible differences that might be related to some of the RBC abnormalities which accompany the disease. The results show that plasma lipids are significantly reduced in patients with sickle cell anemia and that RBC cholesterol was higher in sickle cell patients than in normal subjects.     

Molecular mechanism of cell membrane lipids

The effects of nembutal and ketamine anesthesia on motor evoked potentials (MEPs) and spinal segment reflex (H-response, F and M waves) were investigated in rats by magnetic stimulation. These potentials were generated by magnetic stimulation of the motor cortex and the spinal cord (L4-L5). After application of nembutal, MEP and H-response decreased in amplitude, eventually disappearing. The amplitudes of F and M waves increased and persisted at the increased levels during anesthesia. The latencies of F and M waves were constant before and after anesthesia. Following ketamine administration, the threshold, latency and amplitude of the magnetically induced MEPs, and M, F and H responses were not influenced systematically. The results suggested that MEPs and H-response depression and/or disappearance due to synaptic site suppression after nembutal anesthesia, and the increase and persistence of increased F and M waves amplitudes were all due to the increasing motoneuron excitability, whereas ketamine did not affect synaptic sites subjected to magnetic stimulation.     

Modification of the Ehrlich ascites tumor cell nuclear lipids

The fatty acid composition of Ehrilich ascites tumor cell nuclei was differend when the tumor-bearing mice were fed diets rich in either coconut or sunflower oil. When coconut oil was fed, the monoenoic fatty acid content of many of the nuclear lipids was increased and their polyenoic fatty acid content was reduced as compared with the sunflower oil diet. By contrast, only small changes were produced in the saturated fatty acid contents of the nuclear lipids. The nuclear membrane choline phospholipid, ethanolamine phospholipid and combined serine phospholipid plus inositol phospholipid fractions exhibited statistically significant changes in fatty acid composition, but the sphingomyelins were not altered appreciably by dietary lipid modification. The fatty acid composition of the small quantity of phospholipids associated with the chromatin was much more resistant to diet-induced mosification. Except for sphingomyelin, the fatty acid composition of the chromatin phospholipids was different from that of the corresponding nuclear membrane phospholipids, containing much larger amounts of fatty acids having less than 16 carbon atoms. The fatty acid compositons of the nuclear triaclglycerols and cholesterol esters, which were associated almost entirely with the chromatin, were modified by the dietary lipid modifications. There were no changes in the DNA, RNA or lipid content of these nuclei. Therefore, this experimental system can be used to prepare mamalian nuclei that differ appreciably only in their fatty acyl composition.     

Inhibition in vitro linoleic acid peroxidation and haemolysis by caffeoyltryptophan

Antioxidant activities of caffeoyltryptophan were investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging system, the superoxide anion generation system and the superoxide anion-mediated linoleic acid peroxidation system. At 10 microM, caffeoyltryptophan showed greater scavenging activity on DPPH than dl-alpha-tocopherol or ascorbic acid. DPPH radical scavenging activity of caffeoyltryptophan increased dose-dependently at concentrations ranging from 1 to 50 microM; 1 mol of caffeoyltryptophan reacted with ca 4 mol of radical. Caffeoyltryptophan caused 80% inhibition of superoxide anion generation at 50 microM. The inhibitory activity of caffeoyltryptophan was as strong as that of 5-caffeoylquinic acid. Caffeoyltryptophan inhibited the formation of conjugated diene from linoleic acid. The inhibitory activity increased in the order caffeic acid < 5-caffeoylquinic acid < caffeoyltryptophan < dl-alpha-tocopherol. Effects on the in vitro haemolysis and peroxidation of mouse erythrocytes induced by H2O2 were also examined. Caffeoyltryptophan exhibited strong inhibitory activities; Tryptophan was ineffective in these systems. These data suggest that caffeoyltryptophan may be a natural antioxidant in the human diet and, as such, may intervene in toxicological processes that are mediated by radical mechanisms.     

Modification of ceftibuten transport by changes in lipid fluidity caused by fatty acid glycerol esters

The authors previously observed that schizophrenic patients generated fewer fixations of < or = 50.1 ms in response to faces than did a clinical control group. This study examined whether deficits in short-duration eye movements were related to patients' problems in gestalt perception of faces. Faces were presented in upright and inverted orientations to examine the effects of distorting facial gestalts on eye movements. Normal subjects generated more saccades of < or = 50.1 ms to upright than to inverted faces. Patients' saccades of < or = 50.1 ms did not differ between orientations. Patterns of fixations and of saccades > 50.1 ms did not differ between groups. The results may indicate deficits in these patients in search strategies that underlie perception of facial gestalts.     

The I antigen of human red cell membrane

A high-active I antigen was isolated from human red cells after papainization. Investigations on its chemical composition and its serological properties are reported. 1. The I antigen activity was clearly demonstrated by hemagglutination inhibition studies and by the immuno-double-diffusion with all available anti-I sera. 2. The I antigen did not react with other antibodies directed against red cell antigens thus proving its specificity. Any relationships to antigen activities within the Pr-1/Pr-2, MN, and ABO systems could be excluded. 3. The substance was shown to be a glycoprotein and not a glycolipid. This was confirmed by different delipidation procedures promoting always an increase of I activity. The delipidized material contained only traces of fatty acids, and did not move on thin-layer chromatography in solvent systems normally used for glycolipid development. 4. The I determinant resides on alkali-stable oligosaccharide chains. The main sugars are galactose and N-acetylglucosamine which might be involved in the immunodeterminants.     

Leaching studies of chalcopyrite and sphalerite with hypochlorous acid

Laboratory studies have been conducted on the leaching of chalcopyrite and sphalerite with hypochlorous acid. The effects of stirring speed, temperature, pH, and hypochlorous acid concentration on the leaching rates have been determined. In addition, the leaching mechanisms have been resolved by analyzing the concentrations of the reaction products. It has been found that more than 90 pct extraction of both chalcopyrite and sphalerite can be achieved in one hour using less than 0.3 molar hypochlorous acid at room temperature. The primary leach products of chalcopyrite and sphalerite were sulfur and sulfate in the mole ratios of 1 to 1 and 2 to 1, respectively. A mixed kinetic model was applied to explain the leaching rates of chalcopyrite while a diffusion model was applied to explain the leaching rates of sphalerite. The mixed kinetic model involved steady-state diffusion of hypochlorous acid through the sulfur layer by a chemical reaction at the reaction interface. Good agreement between these models and the leaching rates of both minerals was obtained.     

Interaction of glucose and metformin with isolated red cell membrane

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.     

Dietary menhaden and corn oils and the red blood cell membrane lipid composition and fluidity in hyper- and normocholesterolemic miniature swine

This paper describes the design and synthesis of a tricationic transition state analogue (TSA 1) for the Diels-Alder reaction. TSA 1 contains a bicyclo[2.2.1]heptene ring system that mimics the boat conformation of the Diels-Alder transition state and is designed to bind tightly to antibodies, nucleic acids, and imprinted polymers by means of hydrogen bonds and salt-bridges. This paper also describes the syntheses of the Diels-Alder reaction substrates (diene 2 and dienophile 3) and a sensitive HPLC assay to monitor the formation of Diels-Alder product 4. In contrast to previously reported TSAs and dienophiles for the Diels-Alder reaction that are based upon maleimides, TSA 1 and dienophile 3 are based upon fumaramide. The fumaramide system should destabilize the initially formed boat conformer of Diels-Alder product 4 and stabilize a half-chair conformer. The conversion of the initially formed boat conformer to the half-chair conformer is designed to help prevent Diels-Alder product 4 from binding strongly to catalysts selected to strongly bind TSA 1. This feature should minimize product inhibition, which can be a problem in the catalysis of the Diels-Alder reaction.     

Contribution of whole cell and cytoplasmic polypeptides to apparent red cell membrane alterations

We have compared densitometric tracings of whole cell, cytoplasmic and membrane polypeptide electrophoretic patterns in an attempt to distinguish atypical partitioning from intrinsic membrane polypeptide changes occurring as a result of reticulocyte enrichment, metabolic depletion, N-ethylmaleimide treatment and hereditary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in tary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in the partitioning of polypeptides between membranes and supernatant (cytoplasm) at hemolysis. The power of this approach in resolving the sources of apparent red cell membrane protein alterations is demonstrated in studies with hereditary xerocytes. Suggested altered partitioning of these cells described earlier (Sauberman, N., Fortier, N.L., Fairbanks, G., O'Connor, R.J. and Snyder, L.M. (1979) Biochim. Biophys. Acta 556, 292-313) is further documented and found to be unrelated to the younger cell population or slight metabolic depletion that occurs during the washing of xerocytes prior to hemolysis.     

Oxidation of diclofenac to reactive intermediates by neutrophils, myeloperoxidase, and hypochlorous acid

OBJECTIVE: The experience with MVAC (methotrexate, vinblastine, Adriamycin and cisplatin) chemotherapy in advanced urothelial cancer is reviewed with emphasis on toxicity and efficacy. METHODS: We report on 28 patients with advanced, progressive transitional cell carcinoma (TCC) of the bladder (27) or ureter (1), treated with MVAC. RESULTS: The average number of cycles was 4.5. Leucopenia was the most frequent and severe side effect (18% WHO grade I, 46% GII, 19% GII and 4% GIV). Other side effects were acceptable and could be treated successfully. One patient (complete responder) died of a toxic cause (sepsis), a second patient (partial responder) died of an intestinal bleeding (not drug- or cancer-related). Complete response was seen in 10 patients (36%), partial response and stable disease in 4 patients each (14%), progression in 8 patients (29%), and 2 patients were not evaluable for response. However, relapses were frequent (8 of 12 remaining responders, 66%). Median survival of the whole group was 9 months (0-52), without a significant difference for responders and nonresponders (p = 0.29). CONCLUSION: Our results are comparable to data from the literature with regard to efficacy and toxicity, although detailed toxicity data are unfortunately not always available.     

The effect of cetiedil on red cell membrane permeability

We report the effects of cetiedil, a new antisickling agent, on red cell membrane permeability. With fresh red cells containing normal levels of intracellular ATP, cetiedil increases membrane permeability to both sodium and potassium. With drug concentrations from 100 to 500 microM, net sodium gain exceeds net potassium loss, and the cells quickly swell. Changes are identical with normal and sickle red cells. Membrane permeability returns to normal after washing the cells in buffer free of cetiedil. In the absence of phosphate, ouabain potentiates the cetiedil effect. With external phosphate present, the effect of cetiedil is also enhanced, but ouabain is without effect. Our findings support the idea that the antisickling effect of cetiedil observed in vitro is secondary to cell swelling.     

Existence of a flat phase in red cell membrane skeletons

Biomolecular membranes display rich statistical mechanical behavior. They are classified as liquid in the absence of shear elasticity in the plane of the membrane and tethered (solid) when the neighboring molecules or subunits are connected and the membranes exhibit solid-like elastic behavior in the plane of the membrane. The spectrin skeleton of red blood cells was studied as a model tethered membrane. The static structure factor of the skeletons, measured by small-angle x-ray and light scattering, was fitted with a structure factor predicted with a model calculation. The model describes tethered membrane sheets with free edges in a flat phase, which is a locally rough but globally flat membrane configuration. The fit was good for large scattering vectors. The membrane roughness exponent, zeta, defined through h alpha L zeta, where h is the average amplitude of out-of-plane fluctuations and L is the linear membrane dimension, was determined to be 0.65 +/- 0.10. Computer simulations of model red blood cell skeletons also showed this flat phase. The value for the roughness exponent, which was determined from the scaling properties of membranes of different sizes, was consistent with that from the experiments.     

Spectrin properties and the elasticity of the red blood cell membrane skeleton

Two models of spectrin elasticity are developed and compared to experimental measurements of the red blood cell (RBC) membrane shear modulus through the use of an elastic finite element model of the RBC membrane skeleton. The two molecular models of spectrin are: (i) An entropic spring model of spectrin as a flexible chain. This is a model proposed by several previous authors. (ii) An elastic model of a helical coiled-coil which expands by increasing helical pitch. In previous papers, we have computed the relationship between the stiffness of a single spectrin molecule (K) and the shear modulus of a network (mu), and have shown that this behavior is strongly dependent upon network topology. For realistic network models of the RBC membrane skeleton, we equate mu to micropipette measurements of RBCs and predict K for spectrin that is consistent with the coiled-coli molecular model. The value of spectrin stiffness derived from the entropic molecular model would need to be at least 30 times greater to match the experimental results. Thus, the conclusion of this study is that a helical coiled-coil model for spectrin is more realistic than a purely entropic model.     

Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model

Although the phospholipid composition of the erythrocyte membrane has been studied extensively, it remains an enigma as to how the observed composition arises and is maintained. We show here that the phospholipid composition of the human erythrocyte membrane as a whole, as well as the composition of its individual leaflets, is closely predicted by a model proposing that phospholipid head groups tend to adopt regular, superlattice-like lateral distributions. The phospholipid composition of the erythrocyte membrane from most other mammalian species, as well as of the platelet plasma membrane, also agrees closely with the predictions of the superlattice model. Statistical analyses indicate that the agreement between the observed and predicted compositions is highly significant, thus suggesting that head group superlattices may indeed play a central role in the maintenance of the phospholipid composition of the erythrocyte membrane.     

Study on the relation of human red cell membrane protein band 4.2 to red cell aging--in cases of obstructive jaundice

The relation of human red cell membrane protein band 4.2 to red cell aging both in normal controls and in cases of obstructive jaundice was studied. The studied subjects were 9 people aged 20 to 73 years, the serum bilirubin levels of whom ranged from 4.8 to 26.7mg/dl. And normal controls were 9 people aged 20 to 30 years. First, we separated red cells into several fractions depending on red cell aging by the method of Vettore et al. The analyses of polyacrylamide gel electrophoresis (PAGE) of each RBC membrane sample by Fairbanks' system revealed the results as follows. Primarily, it was observed that in comparison with band 4.1, band 4.2 both in normal controls and in cases of obstructive jaundice increased depending on red cell aging. Secondly, it was suggested that the decrease of band 4.2 protein in cases of obstructive jaundice should not be due to the higher level of serum bilirubin but to the lower level of red cell aging. Because the obstructive jaundice could shorten the life span of red cells and make the average aging level of red cells younger.