The characteristics of the ion exchange mechanisms which regulate volume and pH in fish and amphibian erythrocytes were investigated and compared. Experiments were carried out under steady state conditions and also following adrenergic stimulation both in vivo and in vitro. Under steady state conditions, a decrease in extracellular pH caused an increase in the volume of rainbow trout erythrocytes, and a decrease in the intracellular pH. These pH-induced volume changes were mainly associated with movements of chloride across the chloride/bicarbonate exchange pathway. The sodium/proton exchange mechanism is quiescent at all pH's studied under steady state conditions. Beta adrenergic stimulation of rainbow trout erythrocytes promoted cell swelling and proton extrusion from the erythrocytes. Amiloride inhibited both the volume and pH changes associated with adrenergic stimulation indicating that this response is associated with an increase in the activity of the sodium/proton exchange mechanism on the erythrocyte membrane. The adrenergic swelling and pH responses are enhanced by a decrease in extracellular pH. An increase in bicarbonate concentration reduces the adrenergic pH response, but it is still significant even at 10 mM bicarbonate. DIDS markedly enhanced the beta adrenergic effect on the erythrocyte pH, but abolished the increase in erythrocyte volume. The adrenergic response was independent of temperature between 10 and 18°C. These results support a loosely coupled sodium/proton and chloride/bicarbonate exchange model for the adrenergic response in rainbow trout erythrocytes. The increases in erythrocyte pH and volume following adrenergic stimulation are associated with increases in the haemoglobin:oxygen affinity. The oxygen carrying capacity of the blood is, therefore, increased following adrenergic stimulation in rainbow trout. Carbon dioxide excretion, however, was not significantly affected by adrenergic stimulation. The functional significance of the adrenergic response of fish erythrocytes may be to offset the effects of the Root shift on the oxygen carrying capacity of the blood during acute changes in extracellular pH. In contrast to fish erythrocytes, the sodium/proton exchange mechanism in amphibian erythrocytes is active under steady state conditions. In the presence of bicarbonate movements, this exchange significantly affects the erythrocyte volume, but not the erythrocyte pH. Similar to fish erythrocytes, protons are passively distributed in amphibian erythrocytes under steady state conditions and in Donnan equilibrium with chloride ions. The erythrocyte volume also increases with decreases in extracellular pH as in fish erythrocytes, due to changes in the chloride distribution across the erythrocyte membrane. Adrenergic stimulation does not affect the volume or pH of amphibian erythrocytes either in vivo or in vitro. These animals, therefore, do not appear to regulate erythrocyte pH adrenergically. Amphibians are able to efficiently utilize oxygen stores via both central and peripheral shunting. In addition, the blood of these animals does not have a Root shift. Adrenergic regulation of erythrocyte pH in order to enhance oxygen transport during fluctuations in ambient and internal gas tensions, therefore, is probably less important than it would be in fish. / Science, Faculty of / Zoology, Department of / Graduate

The mechanism of regulation of the Ca²⁺ pump ATPase of the human erythrocyte membrane by calmodulin, cyclic AMP and the anion channel was studied using membrane fragments, resealed "ghosts", inside-out vesicles and a Triton X-100 solubilized enzyme preparation. The (Ca²⁺ + Mg²⁺ )-ATPase activity in erythrocyte membranes or a Triton X-100 solubilized enzyme preparation showed biphasic (high and low affinity) Ca²⁺ activation kinetics. The anionic calcium binding protein, calmodulin, increased both the calcium sensitivity (Kca²⁺) and the maximum velocity (Vmax ) of the enzyme. Certain polyanionic agents (poly-L-aspartic acid, poly-L-glutamic acid), alicyclic sulfonic acids (HEPES,N-2-hydroxyethylpiperazine-N¹-2-ethanesulfonic acid, MES,2-N- (morpholinoethanesulfonic acid)), and aromatic carboxylic acids (benzoic and salicylic acids) increased the Kca²⁺ but not the Vmax of (Ca²⁺ + Mg²⁺ )-ATPase in erythrocyte membranes and Triton X-100 solubilized enzyme preparations. Trifluoperazine (30 μM) antagonized activation of the enzyme by calmodulin and poly-L-aspartic acid, but not by sodium-HEPES or sodium-MES. Limited trypsin proteolysis of (Ca²⁺ + Mg²⁺ )-ATPase in the erythrocyte membrane abolished activation by calmodulin, poly-L-aspartic acid and sodium-HEPES. These results suggest that the modulation of the Ca²⁺ sensitivity of (Ca²⁺ + Mg²⁺ )-ATPase by calmodulin may be associated with the anionic properties of this protein, and that this property can be mimicked by some other anions, probably by interacting at an anion-regulatory site on the enzyme. Cyclic AMP (5 μM) was found to inhibit the (Ca²⁺ + Mg²⁺)-ATPase activity (approx. 20%) in erythrocyte membranes, probably via endogenous cyclic AMP protein kinase, since this effect could be blocked by cyclic AMP protein kinase inhibitor (PKI) from the rabbit skeletal muscle, By contrast, bovine heart PKI stimulated (Ca²⁺ + Mg²⁺ )-ATPase activity (approx. 100%) by increasing the Kca²⁺ but not the Vmax of the enzyme in membrane or Triton X-100 solubilized preparations. At a low calcium concentration the stimulation by bovine heart PKI and saturating levels of calmodulin was additive, suggesting that the two effectors acted by distinct mechanisms. The stimulation of (Ca²⁺ + Mg²⁺ )-ATPase activity by bovine heart PKI was not solely due to its antagonism of the protein kinase because a) modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP protein kinase, but had no effect on the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase; b) trifluoperazine (20 μM) antagonized the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. It is suggested that different mechanisms are involved in the inhibition of cyclic AMP protein kinase and the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase by bovine heart cyclic AMP PKI. Next, the role of anion channel blockers on the (Ca²⁺ + Mg²⁺ )- ATPase was studied. The photolabeling reagent N-(4-azido-2-nitrophenyl)- 2 aminoethylsulfonate (NAP-taurine) was found to inhibit the (Ca²⁺+ Mg²⁺ )-ATPase of fragmented red cell membranes. Half maximal inhibition occurred between 25 μM and 50 μM. At these concentrations Mg²⁺ -ATPase and (Na⁺ + K⁺)-ATPase activities in the membranes were not affected. The reversible inhibition of (Ca²⁺ + Mg²⁺ )-ATPase produced by NAP-taurine in the dark became irreversible after photolysis in the presence of this reagent. Incubation of the membranes with Ca²⁺ , Mg²⁺ , ATP or calmodulin, prior to photolysis in the presence of NAP-taurine, did not protect the enzyme from Inhibition. Limited trypsin proteolysis of (Ca²⁺ + Mg²⁺ )-ATPase in fragmented membranes, which abolished activation by calmodulin, did not affect the inhibition by NAP-taurine. NAP-taurine was found to Inhibit the (Ca²⁺ + Mg²⁺ )-ATPase activity from the cytoplasmic side of the membrane, as determined from the following experiments. Addition of NAP-taurine (50 μM) to resealed erythrocyte ghosts inhibited less than 5% of the (Ca²⁺ + Mg²⁺ )-ATPase activity, compared to 50-60% Inhibition in ghosts resealed in the presence of 50 μM NAP-taurine. Furthermore, NAP-taurine inhibited ATP-dependent Ca²⁺ - transport into inside-out vesicles at a similar concentration (50 μM). The inhibition of the (Ca²⁺ + Mg²⁺ )-ATPase activity of membranes by NAP-taurine appeared to be a direct action on the enzyme, rather than through inhibition of the anion channel, as (Ca²⁺ + Mg²⁺ )-ATPase activity was not inhibited in membranes made from red blood cells reacted irreversibly with 50 μM NAP-taurine or the anion channel blocker 4,4'-diisothiocyano- 2,2' stilbene disulfonate (DIDS) (5 μM) or in membranes assayed in the presence of another anion channel blocker, probenecid (125 μM). This is the first reported selective antagonist of the Ca²⁺ pump, and it is suggested that NAP-taurine could be a useful tool for studying the Ca²⁺- transport ATPase in a variety of cells. / Pharmaceutical Sciences, Faculty of / Graduate

Orientadores: Eneida de Paula, Cleyton Crepaldi Domingues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:11:37Z (GMT). No. of bitstreams: 1 Casadei_BrunaRenata_M.pdf: 5692711 bytes, checksum: 9dcfea152b622eb3bd7d81b793794e3e (MD5) Previous issue date: 2011 / Resumo: A visão atual sobre membranas biológicas abarca descrições cada vez mais complexas devido, principalmente, à descoberta de novos papéis atribuídos aos lipídios e sua heterogeneidade. Em particular a associação de esfingolipídios e colesterol, acrescida de proteínas específicas, constitui a base da formação de domínios membranares conhecidos como lipid rafts. Rafts são microdomínios funcionais de biomembranas e estão envolvidos em diversos processos biológicos como reconhecimento celular, endocitose, transdução de sinal, entre outros processos. Uma estratégia experimental para estudar esses domínios é a preparação de frações de membrana parcialmente resistentes ao tratamento com detergentes, a baixa temperatura (4°C). Neste trabalho demonstramos pela primeira vez o preparo e caracterização de frações resistentes a detergente (DRMs) extraídas de membranas de eritrócito humano, a partir do tratamento com os detergentes não iônicos polioxietileno 20-oleil éter (Brij 98) e polioxietileno 20-cetil éter (Brij 58), seguida de separação por ultracentrifugação em gradiente de sacarose. Essas DRMs foram obtidas a 4°C e 37°C, a partir de membranas intactas e com conteúdo reduzido de colesterol (após tratamento com metil-beta-ciclodextrina) e foram comparadas com DRMs de Triton X-100 (TX-100) em relação ao tamanho, conteúdo proteico e lipídico e grau de organização da bicamada. As frações de DRM de Brij mostraram-se enriquecidas em colesterol e fosfolipídios com ácidos graxos de cadeia saturada (em especial ácido lignocérico das esfingomielinas), características consistentes com lipid rafts. No entanto, DRMs de TX-100 apresentaram maior proporção de esfingomielinas/glicerofosfolipídios que as frações obtidas com Brij, além de menor proporção de fosfatidiletanolamina, um lipídio preferencial da monocamada interna da membrana de eritrócitos. Em relação à solubilização proteica, a membrana do eritrócito foi mais resistente ao tratamento com Brij 98 do que aos outros dois detergentes. Flotilina-2 e estomatina foram encontradas nas frações de DRMs de Brij 98 e Brij 58, porém a redução do conteúdo de colesterol da membrana eritrocitária resultou numa menor associação da flotilina-2 àquelas DRMs. Resultados de Ressonância Paramagnética Eletrônica, com uso de marcadores de spin do tipo doxil-estearato não acusaram variação significativa no grau de empacotamento dos lipídios nas bicamadas de DRMs de Brij 98 e Brij 58 em relação à membrana eritrocitária, diferentemente do observado em DRMs de TX-100 e do que seria esperado para domínios lipídicos na fase líquido-ordenada. Em conclusão, DRMs de eritrócitos humanos, com características compatíveis aos domínios funcionais de biomembranas (rafts), foram obtidas tanto a 4ºC quanto a 37ºC; essas frações resistentes aos Brij apresentaram tamanho, composição proteica e lipídica e grau de empacotamento da bicamada diferente das DRMs de TX-100, indicando um processo de extração diferencial dos componentes da membrana eritrocitária induzida por esses detergentes / Abstract: Depiction of biological membranes has been turning more and more complex lately due to the new roles assigned to their lipid components and their heterogeneity. The association between sphingolipids and cholesterol is the basis of lipid rafts formation. Rafts are functional microdomains of biological membranes which have been associated to different biological processes such as cellular recognition, endocytosis and signal transduction, among others. An useful experimental approach to study lipid rafts is the preparation of membrane fractions partially resistant to detergents under low temperature (4ºC). In this work we report the isolation of detergent resistant membranes (DRMs) from human erythrocytes treated with the non-ionic detergents polioxyethylene 20-oleoyl ether (Brij 98) and polioxyethylene 20-cetyl ether (Brij 58) followed by sucrose gradient ultracentrifugation. Such DRMs were obtained at 4°C and 37°C, from cholesterol-depleted (treated with methyl-beta-cyclodextrin) and intact erythrocyte membranes and they compared to DRMs obtained with Triton X-100 (TX-100) regarding the size, protein and lipid content and membrane fluidity. Brij DRMs were found to be enriched in cholesterol and phospholipids containing saturated fatty acids (especially those from sphingomyelin), features commonly associated to lipid rafts. Nevertheless, TX-100 DRMs presented higher sphingomyelin/phospholipid ratios and lower phosphatidylethanolamine content (a glycerophospholipid mainly present in the inner leaflet) than the Brij's. As for protein solubilization, erythrocyte membranes were more resistant to Brij 98 than to the other two detergents. Flotillin-2 and stomatin were found in both Brij 98 and Brij 8 DRMs. However, flotillin-2 was partially solubilized when DRMs were prepared from cholesterol-depleted erythrocyte membrane. Electron paramagnetic resonance experiments, with doxyl stearic acid spin labels incorporated in the DRMs showed no significant changes in the bilayer compactness of Brij 98 and 58 DRMs in comparison to intact membrane, a unexpected result for lipid domains existing in a liquid-ordered phase, and in contrast to results previously observed with TX-100 DRMs. Altogether, these results show the isolation of DRMs from human erythrocytes treated with Brij 98 and Brij 58 at low (4°C) and physiological temperature (37°C). These detergent-resistant membrane fractions, obtained with Brij 98 and 58 presented some lipid rafts features, although with differences in protein and lipid contents, size and membrane fluidity in comparison to TX-100 DRMs. Besides, these results suggest that TX-100, Brij 98 and Brij 58 induced a different solubilization process on the erythrocyte membrane / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Diabetes mellitus has emerged as a major public health problem with pandemic growth as the International Diabetes Federation estimates that there were 415 million diabetics in 2015 with that number reaching 642 million by 2040, affecting all regions of the world. Globally we are all interconnected when we deal with problems of climate change, water shortage, HIV or Ebola. The war against type 2 diabetes and other non-communicable diseases should be no different, as effective solutions will need expanded global engagement in science to win it. The risk of cardiovascular events in type 2 diabetes remains unchanged despite good control of diabetes and other cardiovascular risk factors. A better understanding of thrombogenicity in diabetes may help to identify novel therapeutic agents and a starting point would be to identify ultrastructural changes in diabetic erythrocytes, platelets and fibrin networks. In diabetes, thrombogenicity is enhanced and is characterised by: hyperactive platelets, higher levels of clotting factors and impaired fibrinolysis. Thus, in this research study, the technique of scanning electron microscopy (SEM) was used to identify ultrastructural abnormalities in erythrocytes, platelets and fibrin networks of diabetic subjects. Distinct abnormal morphological findings were observed in the erythrocytes, platelets and fibrin fibres of diabetic subjects in comparison to the controls. Physiological parameters such as platelet markers and tissue factor levels were also assessed. Flow cytometric analysis revealed hyperactive platelets in the diabetic subjects. The measurement of tissue factor in plasma was completed by using an ELISA. Tissue factor levels in the diabetic subjects were markedly elevated when compared to controls. Biomedical research has provided evidence that has led to the hypothesis that inflammation is the culprit behind almost most chronic illnesses. Hyperglycaemia, a key feature of diabetes, is known to promote a state of low-grade chronic inflammation. A natural method that can resolve acute and chronic inflammation is earthing. Earthing involves coupling your body to the Earth's surface energies by simply walking barefoot or being connected to a conductive device. When earthed, the electrons are conducted into the human body at the same electrical potential as the earth. It is also suggested that free electrons from the earth neutralize the positively charged free radicals that are the hallmark of chronic inflammation. In this study, earthing was accomplished with conductive adhesive patches placed on the sole of each foot and palm of each hand. An earthing cord was connected to the patches and led outdoors to be connected to a stainless-steel rod driven into the ground. Diabetic subjects were earthed for a session of two hours. Bloods were drawn before and just prior to the end of the two-hour session. Morphological SEM findings of the erythrocytes, platelets and fibrin networks at two-hours showed a remarkable difference when compared to findings at baseline. More importantly, the erythrocytes, platelets and fibrin findings revealed that they all almost reverted to looking like control erythrocytes, platelets and fibrin networks. It remains to be seen if earthing will reduce cardiovascular events in diabetics by improving morphology of cells involved in coagulation. / Thesis (PhD)--University of Pretoria, 2016. / Physiology / PhD / Unrestricted

The present kinetic study of the permeability of rabbit erythrocytes has established that carrier systems are involved in the penetration of certain non-electrolytes. Saturation, competitive inhibition, and butanol inhibition kinetics were used to demonstrate the presence of carrier systems and the values of half-saturation constants (ø) were determined for the following water soluble non-electrolytes: glycerol, ethylene glycol, urea, and thiourea. These non-electrolytes are commonly used in permeability studies because they are relatively non-toxic and their small sizes allow penetration of the erythrocyte membrane within a reasonable length of time.

A novel experimental approach for micro-absorption spectroscopy and high-pressure microscopy of single cells is developed and applied to the investigation of morphological, volume, and spectroscopic changes in healthy red blood cells (RBCs) and erythrocytes infected with the malaria parasite Plasmodium falciparum. Through real-time optical imaging of individual erythrocytes (size ~ 7[micrometer]) we determine the change in volume over the pressure range from 0.1 to 210 MPa. The lateral diameter of healthy RBCs decreases reversibly with pressure with an approximate slope of 0.015 [micrometer] / MPa. In infected cells, clear differences in the deformability and between the compression and decompression curves are observed. The results are discussed with respect to the elasticity of the phospholipid membrane and the spectrin molecular network. Employing micro-absorption spectroscopy with spatial resolution of 1.4 [micrometer] in the lateral and 3.6 [micrometer] in the axial direction the visible absorption spectrum of hemoglobin in a single red blood cell is measured under physiological conditions. The spectra of cells infected with the malaria parasite show changes in peak positions and relative intensities in the Soret and [alpha]- and [beta]- bands. These indicate hemoglobin degradation that can be correlated with the stages of the parasite multiplication cycle and can be used as a potential diagnostic marker. The research is further extended towards the understanding of pressure effects on the ligand binding kinetics to heme proteins. For a well characterized reaction at ambient pressure, CO binding to myoglobin in solution, we investigate the transient absorption following laser flash photolysis over eight decades in time at variable pressure and temperature. The data demonstrate that pressure significantly affects the amplitudes (not just the rates) of the component processes. The amplitude of the geminate process increases with pressure corresponding to a smaller escape fraction of ligands into the solvent and a smaller inner barrier.

This research investigates the role of ABO blood type of erythrocytes in their dielectrophoretic response. The dielectrophoresis of erythrocytes of positive ABO blood types was studied at 5 V (peak to peak) and 1 MHz frequency AC field. The study revealed that the ABO blood type had an influence on the dielectrophoretic motion of the erythrocytes, particularly separating AB+ and O+ blood types. This is of particular significance since AB+ is a universal acceptor and O+ is a universal donor for blood transfusion purposes. The influence of field parameters, namely field strength and frequency of the AC field, was also studied for erythrocytes of positive ABO blood types. This research revealed that erythrocytes of each blood type respond differently at various frequencies and field strengths.

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No description available.