Relación entre la presión arterial y el control de la hemorragia post-exodoncia en pacientes bajo tratamiento con warfarina / Relationship between blood pressure and hemorrhage control post exodontia in patients under warfarin treatment

Objetivo: Determinar la relación entre el nivel de pre- sión arterial (PA) y la hemorragia post-exodoncia aplicando medidas de hemostasia local en pacientes bajo tratamiento con warfarina. Materiales y métodos: Este estudio se realizó sobre 30 pacientes (15 hombres y 15 mujeres) bajo tratamiento anti- coagulante oral (TACO) con warfarina. Los pacientes concu- rrían al programa de TACO del Hospital y Centro de Referen- cia de Salud El Pino (HEP y CRS). Se les realizaron una o dos extracciones dentales (n=38) sin suspensión del anticoagulan- te oral a pacientes que tuvieran un coeficiente internacional normalizado (INR) del día menor o igual a 3. Se aplicaron medidas de hemostasia local con gasa compresiva y/o sutura en 30 de las extracciones dentales. Los procedimientos quirúr- gicos fueron llevados a cabo en el Servicio Dental del CRS y HEP. Se registraron las siguientes variables: 1) PA previa a la exodoncia, 2) PA a los 30 minutos, 3) Presencia o ausencia de hemorragia a los 30 minutos post-exodoncia y 4) PA y presen- cia o ausencia de hemorragia a las 24 horas post-exodoncia. Se estudió la relación entre el nivel de PA y la hemorragia post-exodoncia. Resultados: De todos los pacientes evaluados, ninguno presentó hemorragia post-exodoncia en los distintos momen- tos de evaluación, independientemente de cuál fuera su PA. No se encontraron efectos de la variable PA ­considerando valores de PA sistólica (PAS) por debajo de 140 mmHg y de PA diastólica (PAD) menores a 90 mmHg- en relación con la hemorragia post-exodoncia. Conclusión: De acuerdo con los resultados obtenidos en este estudio, la presión arterial con PAS <140 mmHg y PAD <90 mmHg no es un factor que influya en el sangrado post-exodoncia en pacientes bajo tratamiento con warfarina con ≤3 (AU)

Aim: To establish the relationship between blood pres- sure (BP) level and post-exodontic hemorrhage by applying local hemostasis measures in patients under warfarin treat- ment. Materials and methods: This study was conducted in 30 patients (15 men and 15 women) under oral anticoagu- lant (OAC) treatment with warfarin. The patients attended the TACO program of the "Hospital y Centro de Referencia de Salud el Pino (HEP y CRS)". One or two dental extractions (n=38) were performed in the patients that had an INR low- er or equal to 3, without suspending the oral anticoagulant treatment, applying local hemostasis measures with compres- sive gauze and/or suture in 30 of the extractions. The surgical procedure was carried out in the Dental Department of the CRS and HEP. The following variables were registered: 1) BP prior to extraction, 2) BP after 30 minutes, 3) presence or absence of hemorrhage after 30 minutes post-exodontia and 4) BP and presence or absence of hemorrhage 24 hours post-exodontia. The relation between BP level and post-exo- dontic bleeding was studied. Results: Considering all the examined patients, none of them presented post-exodontic hemorrhage at any of the dif- ferent moments of evaluation, regardless of their BP level. No effect of the BP variable ­considering a range of systolic BP SBP) below 140 mmHg and a diastolic BP (DBP) under 90 mmHg- was found in relation to post-exodontic hemorrhage. Conclusion: According to the results obtained in this study, blood pressure with SBP <140 mmHg and DBP <90 mmHg is not an influential factor in post-exodontic bleeding in patients under warfarin treatment with ≤3 (AU)

Under pressure: the extraordinary survival of seal lice in the deep sea.

Lice from pinnipeds - sea lions, seals and walruses - are the only insects capable of surviving marine dives. Throughout their evolutionary history, they have adapted to tolerate hypoxia, high salinity, low temperature and, in particular, to tolerate conditions of high hydrostatic pressure. To understand the limits of the capacity of lice to survive during host deep dives, we conducted a series of controlled experiments in the laboratory. We collected lice from elephant seals and submitted the different life stages to high pressure conditions. Lice were first exposed to one of four hydrostatic pressures: 30, 80, 150 or 200 kg cm-2 They were then exposed a second time to higher or lower hydrostatic pressure conditions to test for the impact of the first experience, which could either be deleterious or trigger physiological adaption, allowing them a better tolerance to high pressure. We found that lice from elephant seals can tolerate hydrostatic pressures higher than 200 kg cm-2 (close to 200 atm), which is equivalent to 2000 m depth. Adults exhibited lower recovery times than nymphs after immersion at high hydrostatic pressure. Our findings show that lice have developed unique adaptations to endure extreme marine conditions. We discuss these extreme performances in relation to the morphological characteristics and physiological responses to diving in these insects.

Analysis of the current-voltage characteristics of polymer-based organic light-emitting diodes (OLEDs) deposited by spin coating / Análisis de la característica corriente-voltaje de diodos orgánicos emisores de luz (OLEDs) basados en polímeros depositados por spin coating / Análise da característica corrente-voltagem de diodos orgânicos emissores de luz (OLEDs) baseados em polímeros depositados por spin coating

Polymer-based organic light-emitting diodes (OLEDs) with the structure ITO / PEDOT:PSS / MDMO-PPV / Metal were prepared by spin coating. It is known that electroluminescence of these devices is strongly dependent on the material used as cathode and on the deposition parameters of the polymer electroluminescent layer MDMO-PPV. Objective. In this work the effect of i) the frequency of the spin coater (1000-8000 rpm), ii) the concentration of the MDMO-PPV: Toluene solution, and iii) the material used as cathode (Aluminium or Silver) on the electrical response of the devices, was evaluated through current-voltage (I-V) measurements. Materials and methods. PEDOT:PPS and MDMO-PPV organic layers were deposited by spin coating on ITO substrates, and the OLED structure was completed with cathodes of aluminium and silver. The electric response of the devices was evaluated based on the I-V characteristics. Results. Diodes prepared with thinner organic films allow higher currents at lower voltages; this can be achieved either by increasing the frequency of the spin coater or by using concentrations of MDMO-PPV: Toluene lower than 2% weight. A fit of the experimental data showed that the diodes have two contributions to the current. The first one is attributed to parasitic currents between anode and cathode, and the other one is a parallel current through the organic layer, in which the carrier injection mechanism is mediated by thermionic emission. Conclusions. The results of the fitting and the energy level alignment through the whole structure show that PPV-based OLEDs are unipolar devices, with current mainly attributed to hole transport.

Se fabricaron diodos orgánicos emisores de luz (OLEDs) con la estructura ITO / PEDOT:PSS / MDMO-PPV / Metal mediante la técnica de spin coating. Es ampliamente conocido que la electroluminiscencia de estos diodos depende fuertemente del material usado como cátodo y también de los parámetros de crecimiento de la capa del polímero electroluminiscente MDMO-PPV. Objetivo. En este trabajo el efecto de i) la frecuencia del spin coater (1000-8000 rpm), ii) la concentración de la solución MDMO-PPV: Tolueno y iii) el material usado como cátodo (plata o aluminio) sobre la repuesta eléctrica de los dispositivos, fue evaluado a través de medidas de corriente-voltaje (I-V). Materiales y métodos. Películas delgadas de los materiales orgánicos PEDOT:PSS y MDMO-PPV fueron depositados por spin coating sobre sustratos de ITO, y la estructura del OLED fue terminada con cátodo de plata y aluminio. La respuesta eléctrica de los dispositivos fue evaluada a través de su característica I-V. Resultados. Los diodos fabricados con películas orgánicas más delgadas son los que suministran mayores corrientes a menores voltajes. Esto puede lograrse ya sea incrementando la frecuencia de rotación del spin coating o usando concentraciones de MDMO-PPV: Tolueno menores al 2% en peso. Un ajuste de los datos experimentales demostró que los diodos poseen contribuciones de una corriente parásita entre ánodo y cátodo, y otra corriente paralela en donde el mecanismo predominante de inyección de portadores a la capa orgánica es a través de emisión termoiónica. Conclusiones. El ajuste de los datos experimentales, junto con la posición de niveles de energía a través de la heteroestructura, demuestra que los OLEDs basados en derivados de PPV son dispositivos unipolares, en el que la corriente se atribuye principalmente a transporte de huecos.

Foram fabricados diodos orgânicos emissores de luz (OLEDs) com a estrutura de ITO / PEDOT: PSS / MDMO-PPV / metal, pela técnica de spin coating. É amplamente conhecido que a eletroluminescência destes diodos depende fortemente do material utilizado como cátodo, e também dos parâmetros de crescimento da camada de polímero eletroluminescente MDMO-PPV. Objetivo. Neste trabalho o efeito de i) a freqüência do spin coater (1000-8000 rpm), ii) a concentração da solução MDMO-PPV: Tolueno e iii) o material utilizado como cátodo (prata ou alumínio) sobre a resposta elétrica dos dispositivos, foi avaliado por medidas de corrente-voltagem (I-V). Materiais e métodos. Películas finas de materiais orgânicos PEDOT: PSS e MDMO-PPV foram depositadas por spin coating sobre substratos de ITO e a estrutura do OLED foi terminada com cátodo de prata e de alumínio. A resposta elétrica dos dispositivos foi avaliada pela sua característica I-V. Resultados. Os diodos feitos de películas orgânicas finas fornecem maiores correntes a menores voltagens. Isto pode ser conseguido, quer através do aumento da velocidade de rotação do spin coating ou usando concentrações de MDMO-PPV: Tolueno menores de 2% em peso. Um ajuste dos dados experimentais mostrou que os diodos têm uma contribuição de uma corrente parasita entre anodo e catodo, e outra corrente paralela, onde o principal mecanismo da injeção de portadores da camada orgânica é através da emissão termiônica. Conclusões. O ajuste dos dados experimentais, juntamente com a posição dos níveis de energia através da heteroestrutura, mostra que os OLEDs baseados em derivados de PPV são dispositivos unipolares, onde a corrente é atribuída principalmente ao transporte de ocos.

Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion.

The Plasmodium falciparum ring-erythrocyte surface antigen (RESA)-like putative protein was identified and characterised. PCR and RT-PCR assays revealed that the gene encoding this protein was both present and being transcribed in P. falciparum strain FCB-2 16 h after erythrocyte invasion. Indirect immunofluorescence studies detected this protein in infected erythrocyte (IE) cytosol in dense fluorescent granules similar to Maurer's clefts at 16-20 h (parasites in ring and trophozoite stages) and very strongly on IE membranes at 22 h, suggesting that it is synthesised during early ring stages (16 h) and transported to the infected red blood cell (RBC) membrane surface during the trophozoite stage (22 h). Western blotting showed that antisera produced against polymerised synthetic peptides of this protein recognised a 72-kDa band in P. falciparum schizont lysate. P. falciparum RESA-like peptides used in normal RBC binding assays revealed that peptides 30326 ((101)NAEKI LGFDD KNILE ALDLFY(120)), 30334 ((281)RVTWK KLRTK MIKAL KKSLTY(300)) and 30342 ((431)SSPQR LKFTA GGGFC GKLRNY(450)) bind with high activity and saturability, presenting nM affinity constants. These peptides contain alpha-helical structural elements, as determined by circular dichroism, and inhibit P. falciparum in vitro invasion of normal RBCs by up to 91%, suggesting that some RESA-like protein regions are involved in intra-erythrocyte stage P. falciparum invasion.

Biomarkers and bioindicators of the health condition of Ameca splendens and Goodea atripinnis (Pisces: Goodeaidae) in the Ameca River, Mexico.

Pollution of water bodies causes stress on organisms inhabiting them. Determination of biomarkers and bioindicators on fish populations reflects whether they are subject to stress. We assessed two populations of Ameca splendens and Goodea atripinnis in a reference site (spring "El Rincon") and De La Vega reservoir, which receives wastewater of a sugar industry, on Ameca River course. We analyzed level of lipid peroxidation and enzymatic activities of gamma-glutamyl-transpeptidase, acetylcholinesterase and ethoxyresorufin-O-deethylase as biomarkers; we also studied age classes and various body indexes. Environmental factors were recorded and a water quality index was assessed. Water quality was better in the spring than in the reservoir. Organisms inhabiting the reservoir presented higher oxidative stress by the lipid peroxidation levels, and neurotoxic impacts by the acetylcholinesterase and some detoxification mechanisms were evident by the gamma-glutamyl-transpeptidase and ethoxyresorufin-O-deethylase activities. Integrated Biomarker Response demonstrate that De La Vega reservoir is a more stressing place to organisms living there, particularly for A. splendens. In both species, males were more affected than females. Condition and reproductive parameters in reservoir showed evidences of physiological changes due to xenobiotics exposure and suggest a tactic of the organisms to survive in this site. Both biomarkers and body indexes revealed that A. splendens is a more sensitive species than G. atripinnis to environmental stress.

Plasmodium falciparum merozoite surface protein 6 (MSP-6) derived peptides bind erythrocytes and partially inhibit parasite invasion.

This work shows that Plasmodium falciparum merozoite surface protein-6 (MSP-6) peptides specifically bind to membrane surface receptor on human erythrocytes. Three high activity binding peptides (HABPs) were found: peptides 31175 (41MYNNDKILSKNEVDTNIESN60) and 31178 (101YDIQATYQFPSTSGGNNVIP120) in the amino terminal region and 31191 (361EIDSTINNLVQEMIHLFSNNY380) at the carboxy terminal. Their binding to erythrocytes was saturable. HABPs 31191 and 31178 recognized 56 and 26 kDa receptors on erythrocyte membrane and inhibited in vitro Plasmodium falciparum merozoite invasion of erythrocytes by between 27% and 46% at 200 microg ml(-1) concentration, suggesting that these MSP-6 protein peptides play a possible role in the invasion process.

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes.

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.

Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells.

Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex's strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54-kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an alpha-helical structure. HABP-target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus-target cell interactions and implications for developing strategies for controlling this disease.

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines.

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion.

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells.

Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasite's in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro.

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .

Identifying Plasmodium falciparum merozoite surface protein-10 human erythrocyte specific binding regions.

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein's possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.

Protection against malaria induced by chirally modified Plasmodium falciparum's MSP-1 42 pseudopeptides.

The C-terminal portion of the Plasmodium falciparum blood stage MSP-1 antigen plays a key role in invasion of human erythrocytes. The MSP-1(1282-1301) non-polymorphic 1585 peptide, from the processed MSP-1(42) fragment, is poorly immunogenic and highly alpha-helical [Angew. Chem. Int. Ed. 40 (2001) 4654]. Assessing the alpha-carbon asymmetry and its implication in the host immune response is proposed in this work to overcome the 1585 peptide's immunological properties. Accordingly, the effect of incorporating single D-amino acids and psi-[CH(2)-NH] isoster bonds into the 1585 peptide was examined both at the immunogenic and 3D-structure levels. Therefore, specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with psi-[CH(2)-NH] isoster bonds transforming this molecule into a high specific HLAbeta1*1101 allele binder. D-analog pseudopeptide immunized animals induced antibodies selectively recognizing a recombinant as well as native MSP-1(42) and MSP-1(33) fragments. Protection and low parasitemia levels were induced in Aotus monkeys immunized with the EVLYL(dK)PLAGVYRSLKKQLE analog. Peptide alpha-carbon chiral transformation is therefore an important target for structural modulation and, consequently, represents a novel approach towards designing multi-component subunit-based malarial vaccines.

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides.

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes.

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.

Liver stage antigen 3 Plasmodium falciparum peptides specifically interacting with HepG2 cells.

Binding assays were carried out with 20 amino acid long peptides covering the complete 200-kDa Liver stage antigen (LSA) 3 protein sequence to identify its HepG2 cell binding regions. Seventeen HepG2 cell high-activity binding peptides (HABPs) were identified in the LSA-3 protein. Seven HABPs were found in the nonrepeat (NRA) region A; five of these formed a 100 amino acid long HepG2 cell binding region located between residues 21Ile and 120Thr. Six HABPs were found in the R2 region and another four in the NRB2 region. LSA-3 protein HABPS bound saturably to HepG2 cells having nanomolar affinity constants and bound specifically to 31, 44, and 70 kDa HepG2 cell membrane proteins. Some of them were located in antigenic and immunogenic LSA-3 protein regions. Immunofluorescence and immunoblotting assays using goat sera immunized with LSA-3 protein peptides recognized P. falciparum (FCB-2 strain) erythrocyte stage proteins (58, 68, 72, 81, 86, 160, and 175 kDa). This reactivity was due mainly to the VEESVAEN motif present in some erythrocyte stage proteins. However, our results suggest that antibodies against LSA-3 regions had a crossed reaction with another 86-kDa protein, and that this crossed reaction was due to a motif present in the NRA region.

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum-derived rhoptry-associated protein 1 peptides.

Rhoptry-associated protein 1 (RAP1) is a merozoite antigen within Plasmodium falciparum rhoptries as yet having no specific function described for it. Synthetic peptides spanning the RAP1 sequence were tested in erythrocyte binding assays to identify possible RAP1 functional regions. Five high activity binding peptides (HABPs) were identified; 26201, 26202, 26203 and 26204 spanned residues 461C-K540 within RAP1 Cys region, whilst 26188 (201T-Y220) was located in p67 amino terminal. The results showed that peptide binding was saturable, some HABPs inhibited in vitro merozoite invasion and specifically bound to a 72 kDa protein in red blood cell membrane. HABP possible function in merozoite invasion of erythrocytes is also discussed.

Sporozoite and liver stage antigen Plasmodium falciparum peptides bind specifically to human hepatocytes.

Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell binding assays to identify regions involved in parasite invasion. SALSA 20608 ( 21IWASEKKDEKEASEQGEESHY40) and 20611 ( 64KKDDGTDKVQEKVLEKSPKY83) peptides were determined as having high binding activity in HepG2 cell assays, some of them were located in immunogenic regions. Immune-fluorescence antibody test with 24276 (20608 peptide analogue, CGIWSSMKMDEKMAAMQGEESHCG) showed sporozoite and merozoite reactivity. This data suggests SALSA high activity binding peptides' (HABPs) possible role in hepatic cell invasion and merozoite invasion of erythrocytes.

Identification of Plasmodium falciparum reticulocyte binding protein RBP-2 homologue a and b (PfRBP-2-Ha and -Hb) sequences that specifically bind to erythrocytes.

Plasmodium falciparum reticulocyte binding protein RBP-2 homologues a and b (PfRBP-2-Ha and -Hb) have been described as being high molecular weight proteins, expressed at the P. falciparum merozoite apical extreme, belonging to a family of proteins found in other Plasmodium involved in the search for erythrocyte populations before being invaded by merozoites. 185, 20-mer-long non-overlapping peptides, spanning the entire PfRBP-2-Ha and -Hb sequences, were synthesised, radiolabelled and tested in erythrocyte binding assays. Fifteen PfRBP-2-Ha and -Hb high binding activity peptides (HBAPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants were between 70 and 300 nM and Hill coefficients were 1 approximately. HBAPs residues critical for binding to erythrocytes were determined. Cross-linking was performed allowing possible receptors for PfRBP-2-Ha and -Hb to be identified on the surface of the erythrocytes. Some of the HABPs showed merozoite invasion inhibition greater than 90% in in vitro assays.