Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion.

Protein-protein interactions (IPP) play an essential role in practically all biological processes, including those related to microorganism invasion of their host cells. It has been found that a broad repertoire of receptor-ligand interactions takes place in the binding interphase with host cells in malaria, these being vital interactions for successful parasite invasion. Several trials have been conducted for elucidating the molecular interface of interactions between some Plasmodium falciparum and Plasmodium vivax antigens with receptors on erythrocytes and/or reticulocytes. Structural information concerning these complexes is available; however, deeper analysis is required for correlating structural, functional (binding, invasion, and inhibition), and polymorphism data for elucidating new interaction hotspots to which malaria control methods can be directed. This review describes and discusses recent structural and functional details regarding three relevant interactions during erythrocyte invasion: Duffy-binding protein 1 (DBP1)-Duffy antigen receptor for chemokines (DARC); reticulocyte-binding protein homolog 5 (PfRh5)-basigin, and erythrocyte binding antigen 175 (EBA175)-glycophorin A (GPA).

"Molecular analysis of the rare S-s- red blood cell phenotype in blood donors and patients in south-east Brazil".

BACKGROUND AND OBJECTIVES: The rare S-s- phenotype has two main molecular backgrounds. GYPB deletions give rise to the S-s-U- phenotype, which loses the expression of the U antigen, while variant GYPB alleles usually lead to the S-s-U+var phenotype, which express a variant U antigen. The S-s- phenotype is typically found in people of African origin and represents a challenge in transfusion sets, especially when S-s- patients develop anti-U. Molecular analysis is the most reliable method for determining U antigen status. We studied the molecular basis of the S-s- phenotype in donors and patients at Regional Blood Center of Ribeirão Preto. MATERIAL AND METHODS: Five patients and 25 donors with the S-s- phenotype were investigated through real-time PCR for the GYPB*S/s polymorphism, followed by an allele-specific/RFLP-PCR for GYPB deletion (GYPB*Null) and for its main variants: GYPB*P2 and GYPB*NY. DNA sequencing was conducted in one sample. RESULTS: Two samples were heterozygous GYPB*P2/GYPB*NY, eight were homozygous/hemizygous for GYPB*P2 and 19 samples were homozygous for GYPB*Null. A hybrid gene (GYPB-E-B.Ros) was found in one sample after discrepant results in the initial tests. CONCLUSION: GYPB deletion is the main mechanism responsible for the S-s- phenotype in our donors and patients. It is essential to evaluate the main GYPB variant alleles when genotyping in order to obtain the correct prediction of the phenotype. Hybrid genes lead to discrepancies between genotype and phenotype and may not be detected by conventional molecular assays.

Low expression of stem cell antigen-1 on mouse haematopoietic precursors is associated with erythroid differentiation.

Sca1 is a surface marker of haematopoietic stem cell but its role in erythropoiesis is still largely unknown. In this work we evaluated the ability of Sca1⁺ cells to differentiate into cells of the erythrocytic lineage. We performed FACS analysis of complete and purified Sca1⁺ bone marrow cells from C3H/HeNHsd mice and measured the expression of CD71 and Terr119 to evaluate the stages in erythroid development. Definitive erythropoiesis was evident within the complete bone marrow, while only proerythroblasts were found in Sca1⁺ cells, suggesting that Sca1 is a negative regulator of erythropoiesis. We also used FDCP-mix cells and their PU.1 and SCL transfectants. The PU.1 transfectant showed significantly increased expression of Sca1 and was not induced to differentiate into red blood cells, while the SCL transfectant showed significantly lower expression of Sca1 and produced red blood cells. The results of this study suggest that increased Sca1 expression on erythropoietic precursors inhibits erythroid differentiation.

Hemocompatibility and biofunctionality of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) copolymers.

To mask the antigenic sites of cells for cell therapies, especially for blood transfusion, we investigated the hemocompatibility of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) compared with that of the homopolymer without PEG. Our strategy relies on the potential ability of these copolymers to self-assemble at the erythrocyte surface. The cationic sequence of the copolymer should be able to interact with the glycocalyx by ionic interaction. The other sequence, based on a polyethyleneglycol moiety, should prevent both nonspecific interactions and specific recognition of the biological surface. The hemocompatibility of these copolymers was assessed by analyzing alterations in human erythrocyte membrane viscoelasticity, morphology, granularity, and aggregation. Their properties to mask ABO system and three erythrocyte glycophorin sites were investigated. No alterations in the erythrocyte morphology were observed by confocal microscopy. On the other hand, a partial masking of different specific glycophorin sites leads to future optimization of the macromolecular structures of these functionalized copolymers.

Heme-oxygenases during erythropoiesis in K562 and human bone marrow cells.

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon.

BACKGROUND: Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. METHODS: Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. RESULTS: GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. CONCLUSION: Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

Effect of cholesterol depletion and temperature on the isolation of detergent-resistant membranes from human erythrocytes.

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37 degrees C, or from cholesterol-depleted cells at 4 degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4 degrees C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C(12)E(8) DRMs prepared at 4 degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4 degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs.

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins.

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L. obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca2+ and Mg2+ ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L. obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L. obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation.

Molecular analyses of GYPB in African Brazilians.

The molecular background of variant forms of GYPB is not well studied in Brazilians of African descent. The present study was carried out to determine the molecular bases of the S-s- phenotype and the frequency of GYPB*S silent gene for the S-s+ phenotype in a blood donor population of African Brazilians. In this study, 165 blood samples from African Brazilians (Northeastern Brazil) who phenotyped as S-s- (n = 17) and S-s+ (n = 148) by hemagglutination were selected. Allele-specific (AS)-PCR and PCR-restriction fragment length polymorphism (RFLP) were used to identify the variant forms of GYPB. In 13 of 17 S-s- samples (76.5%), both GYPB were deleted. In 137 of the 148 S-s+ samples (92.6%), the AS-PCR was consistent with the S-s+ phenotype. In 4 of the S-s- samples (23.5%) and 11 of the S-s+ samples (7.4%), the AS-PCR showed the presence of a GYPB*S allele associated with silencing of S. In the 4 donors with the S-s- phenotype, there was homozygosity (or hemizygosity) for the GYP(P2) allele (n = 2), homozygosity (or hemizygosity) for the GYP(NY) allele (n = 1), and heterozygosity for the GYP(P2) and GYP(NY) alleles (n = 1). In the 11 donors with the S-s+ phenotype, there was heterozygosity for GYP(P2) allele (n = 8) and heterozygosity for GYP(NY) allele (n = 3). This study reports for the first time the molecular mechanisms responsible for the S-s- phenotype in a population of African Brazilians and provides new information about the frequency and molecular bases of the GYPB*S silent gene (7.4%) in this population.

Aspectos estruturais da membrana eritrocitária / Structural aspects of the erythrocyte membrane

Este artigo descreve as estruturas e funções da membrana eritrocitária e sua importância na medicina transfusional. A membrana eritrocitária é uma das membranas mais conhecidas em termos de estrutura, função e genética. Como qualquer membrana plasmática, tem como função mediar transportes e, ainda, fornece ao eritrócito resistência e maleabilidade. De acordo com a International Society of Blood Transfusion (ISBT), são mais de 500 antígenos expressos na membrana das hemácias e, destes, cerca de 270 estão envolvidos nos casos de reação transfusional e doença hemolítica do feto e do recém-nascido. Na classificação feita pela ISBT, destaca-se a série de alta freqüência representada por antígenos presentes em mais de 99 por cento dos indivíduos de uma população. Estes antígenos são conhecidos também como antígenos públicos e a maioria, quando ausente, determina problemas graves do ponto de vista transfusional. Como exemplo dessa problemática, uma gestante com ausência do antígeno P já sofreu seis abortos de repetição por insuficiência placentária devido ao anticorpo formado pela ausência do antígeno. Proteínas importantes são descritas nesta revisão como: banda 3, glicoforinas, espectrina e outras. A banda 3 é a mais abundante proteína integral da membrana do eritrócito e sua principal função é mediar a troca de cloro e ânions de bicarbonato através da membrana plasmática. A segunda proteína integral mais abundante é a sialoglicoproteína glicoforina A (GPA). Com um alto conteúdo de ácido siálico, a GPA contribui com a rede de carga negativa na superfície da membrana do eritrócito, minimizando, assim, a interação célula-célula e prevenindo sua agregação. Glicoforina C (GPC) é o receptor para PfEBP-2 (baebl, EBA-140), o mais novo local de ligação identificado para o Plasmodium falciparum.O complexo terciário - espectrina, actina e 4.1R - define a rede de citoesqueleto da membrana do eritrócito e é ainda responsável pela estabilidade sob mecanismos...

This article describes the structures and functions of the erythrocyte membrane and its importance in transfusional medicine. The erythrocyte membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any other plasma membrane, it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. According to the International Society of Blood Transfusion (ISBT), more than 500 antigens are expressed in the erythrocyte membrane, and around 270 are involved in transfusion reaction cases and hemolytic diseases of the fetus and newborn. In the ISBT classification, the high frequency series is represented by antigens in more than 99 percent of population (high prevalence antigen). In transfusion, the absence of these antigens determines severe problems as for example, one woman without the P antigen suffered 6 repetitive miscarriages due to placental insufficiency, which was caused by an antibody formed against the absent P antigen. Some important erythrocyte membrane proteins are described here including Band 3, Glycophorins and spectrin. The most abundant integral membrane protein is Band 3 and its main function is to mediate exchange of chloride and bicarbonate anions across the plasma membrane. The second most abundant integral membrane protein in the human erythrocyte is sialoglycoprotein glycophorin A (GPA). With its high sialic acid content, GPA is the main contributor to the net negative cell-surface charge and is thus critical for minimizing cell-cell interactions and preventing red cell aggregation. Glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. The ternary complex of spectrin, actin and 4.1R defines the nodes of the erythrocyte membrane skeletal network, and is inseparable from membrane stability when under mechanical stress. This erythrocyte membrane review...

A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry.

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.

Admixture in Hispanics: distribution of ancestral population contributions in the Continental United States.

The effect of gene flow on Hispanic populations from different geographic regions of the United States was analyzed using six autosomal DNA markers (LDLR, GYPA, HBGG, D7S8, GC, and HLA-DQA). By region of sampling, the Hispanic populations showed different ancestry contributions, from a trihybrid structure with European, Native American, and African contributions (California, Nevada, Florida, New Jersey, and Virginia) to a dihybrid structure with European and American contributions (Southwest population) or European and African contributions (Pennsylvania and Southeast population). These findings allowed us to define two regional groups, the West and the East. In the former, Native American contributions ranged from 35.58% to 57.87%; in the East region the values ranged from 0% to 21.27%. An African influence was similar in both regions, ranging from 0% to 17.11%, with a tendency of increasing in the East region. These data reflect the different origins of the Hispanic populations that led to the present ones. In the West, Hispanics are mostly of Mexican origin, and in the East, they are predominantly of Cuban and Puerto Rican origin.

Adhesión eritrocitaria: método aplicado a patologías asociadas con proteínas portadoras de grupos sanguíneos eritrocitarios / Red blood cell adhesion. A method to be applied to the pathologies associated with protein carring erythrocitic blood groups

El estudio de la agregación eritrocitaria cobra importancia en la medida que permita cuantificar anormalidades circulatorias en diversas patologías. En condiciones normales los glóbulos rojos (GR) se agregan en estructuras cilíndricas denominadas rouleaux, mientras que en condiciones anormales, los agregados adoptan formas irregulares con tendencia morfológica esferoidal. Estas formas anómalas pueden ser inducidas por alteraciones celulares (disminución del contenido de ácido siálico de las glicoproteínas), o por factores extracelulares. El objetivo de éste trabajo fue estudiar la adhesión eritrocitaria relacionada con patologías asociadas a glicoforina A portadora de los antígenos MN de grupos sanguíneos eritrocitarios mediante metodología no convencional. Se estudió la morfología de los agregados eritrocitarios de 36 pacientes diabéticos, 20 pacientes hipertensos, 40 individuos sanos y GR con tratamiento enzimático a distintos tiempos. La agregación se cuantificó con un parámetro de forma de los agregados, denominado ASP (Aggregate Shape Parameter), definido como la relación entre el área proyectada de los agregados y el cuadrado de su perímetro. Los datos fueron obtenidos con muestras de suspensiones de GR en plasma autálogo, al 2 por ciento de hematocrito, las cuales fueron observadas con una cámara CCD (Camera Coupled Digital) y procesadas numéricamente con un procesador digital de imágenes (DIP), ambos acoplados a un microscopio invertido. Las glicoforinas (GP) portadoras de los antígenos M y N, juegan un papel estructural en la preservación de la forma del GR. Esto está ligado a la gran cantidad de ácido siálico (AS) que contienen, el que confiere al eritrocito, una carga negativa. Al aumentar el tiempo de incubación de la tripsina con los GR se produce una desialización progresiva de las GP de la membrana. Como consecuencia de ello se observa la formación de clusters (agregados irregulares) que llevan a valores progresivamente aumentados del ASP. Los pacientes diabéticos (ASP = 0,65 ñ 0,18) e hipertensos (ASP = 0,69 ñ 0,19) presentan valores significativamente mayores (p<10-5) que los controles normales (ASP = 0,28 ñ 0,15). Un aumento de la agregación eritrocitaria representa un importante factor de riesgo en el desarrollo de patologías vasculares, con posible deterioro de la microcirculación. En éste sentido, el ASP provee una útil referencia para cuantificar alteraciones en la morfología de los agregados

Adhesión eritrocitaria: método aplicado a patologías asociadas con proteínas portadoras de grupos sanguíneos eritrocitarios / Red blood cell adhesion. A method to be applied to the pathologies associated with protein carring erythrocitic blood groups

El estudio de la agregación eritrocitaria cobra importancia en la medida que permita cuantificar anormalidades circulatorias en diversas patologías. En condiciones normales los glóbulos rojos (GR) se agregan en estructuras cilíndricas denominadas rouleaux, mientras que en condiciones anormales, los agregados adoptan formas irregulares con tendencia morfológica esferoidal. Estas formas anómalas pueden ser inducidas por alteraciones celulares (disminución del contenido de ácido siálico de las glicoproteínas), o por factores extracelulares. El objetivo de éste trabajo fue estudiar la adhesión eritrocitaria relacionada con patologías asociadas a glicoforina A portadora de los antígenos MN de grupos sanguíneos eritrocitarios mediante metodología no convencional. Se estudió la morfología de los agregados eritrocitarios de 36 pacientes diabéticos, 20 pacientes hipertensos, 40 individuos sanos y GR con tratamiento enzimático a distintos tiempos. La agregación se cuantificó con un parámetro de forma de los agregados, denominado ASP (Aggregate Shape Parameter), definido como la relación entre el área proyectada de los agregados y el cuadrado de su perímetro. Los datos fueron obtenidos con muestras de suspensiones de GR en plasma autálogo, al 2 por ciento de hematocrito, las cuales fueron observadas con una cámara CCD (Camera Coupled Digital) y procesadas numéricamente con un procesador digital de imágenes (DIP), ambos acoplados a un microscopio invertido. Las glicoforinas (GP) portadoras de los antígenos M y N, juegan un papel estructural en la preservación de la forma del GR. Esto está ligado a la gran cantidad de ácido siálico (AS) que contienen, el que confiere al eritrocito, una carga negativa. Al aumentar el tiempo de incubación de la tripsina con los GR se produce una desialización progresiva de las GP de la membrana. Como consecuencia de ello se observa la formación de clusters (agregados irregulares) que llevan a valores progresivamente aumentados del ASP. Los pacientes diabéticos (ASP = 0,65 ñ 0,18) e hipertensos (ASP = 0,69 ñ 0,19) presentan valores significativamente mayores (p<10-5) que los controles normales (ASP = 0,28 ñ 0,15). Un aumento de la agregación eritrocitaria representa un importante factor de riesgo en el desarrollo de patologías vasculares, con posible deterioro de la microcirculación. En éste sentido, el ASP provee una útil referencia para cuantificar alteraciones en la morfología de los agregados (AU)

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases.

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD- and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.