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L Pereira da Silva

Publications and source records attributed to L Pereira da Silva.

At least 19 recordsLinked to original sources
PubMed2003

Amperometric sensor for glutathione reductase activity determination in erythrocyte hemolysate.

The development of an amperometric sensor for glutathione reductase (GR) activity in erythrocyte hemolysate to contribute to oxidative stress evaluation is presented. In this assay, the reduced form of glutathione, the product of the GR reaction, reacts with 5,5(')-dithiobis(2-nitrobenzoic acid), producing GSTNB, which is easily reduced in the electrode surface. The current was recorded during 180 s after the sample addition, applying a potential of -300 mV. The sensor presented a suitable sensitivity, a good operational range, and precision. The effects of pH variations and specific uncompetitive inhibitor (safranin-O) in the enzyme activity were also evaluated. The GR activity determination in human erythrocyte hemolysate using this method has provided results that are statistically equal to those obtained by the classical spectrophotometric method, with 95% of confidence. The advantages of this method are the saved time, reagents, and samples and the possibility of its use in the field.

Cell Extracts↗
PubMed1997

Plasmodium falciparum asparagine and aspartate rich protein 2 is an evolutionary conserved protein whose repeats identify a new family of parasite antigens.

We describe here a new Plasmodium falciparum antigen, asparagine and aspartate rich protein 2 (PfAARP2) of 150 kDa, which is encoded by a unique gene on chromosome 1. PfAARP2 is first expressed 12 h post-invasion and accumulates in trophozoites and schizonts. Immunofluorescence studies indicate that PfAARP2 is translocated into the red blood cell cytoplasm. The central region of Pfaarp2 contains blocks of repetitions encoding asparagine and aspartate residues, which define a new family of related genes dispersed on different chromosomes, and two members of this family have also been identified. Interestingly, the non-repeated N- and C-termini of PfAARP2 display significant similarity to two yeast and human predicted proteins, and its possible function is discussed.

Amino Acid Sequence↗
PubMed1997

Plasmodium falciparum AARP1, a giant protein containing repeated motifs rich in asparagine and aspartate residues, is associated with the infected erythrocyte membrane.

During Plasmodium falciparum asexual intraerythrocytic development, the host's cell plasma membrane is modified by the insertion of parasite proteins. One or more of these modifications mediate the cytoadherence of infected erythrocytes to host vascular endothelium. However, these surface antigens can be the target of cytophilic antibodies which promote phagocytosis of the infected erythrocyte. It has been proposed that antibodies directed to epitopes rich in asparagine play an important role in this process, which has promoted efforts to isolate the corresponding gene(s). We describe here P. falciparum asparagine- and aspartate-rich protein 1 (PfAARP1), a new giant (circa 700-kDa) protein associated with the infected erythrocyte membrane which is rich in asparagine and aspartate residues due to the presence of nine blocks of repeats. Topology analysis predicts that PfAARP1 has multiple transmembrane domains and at least five external loops. Human antibodies immunopurified against a sequence composed exclusively of asparagine and aspartate amino acids derived from PfAARP1 label the surface of the infected erythrocyte, demonstrating that such motifs are exposed. Interestingly, external loop 4 of PfAARP1 contains repetitions of these residues, and their possible role as a target of cytophilic antibodies is discussed.

Amino Acid Sequence↗
PubMed1995

Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected by Plasmodium falciparum.

The immunofluorescence detection of parasite-specific antigens on the surface of red blood cells infected by Plasmodium falciparum parasites is usually performed by visual detection under a fluorescence microscope. We describe here a technique permitting the analysis of surface immunofluorescence labelling by flow cytometry. Infected red blood cells are selected on the basis of their parasitic DNA and RNA content by Hoechst and Thiazole Orange vital dyes. Cytometric analysis of these labels, as well as general erythrocyte characteristics assessed by analysis of forward and side scatter allows the selection of viable intact infected erythrocytes from other blood cells. The integrity of these selected erythrocytes was confirmed by the absence of labelling with antibodies directed against internal components such as spectrin. This technique permits the detection of specific surface immunofluorescence staining on red blood cells infected with mature stages of P. falciparum by antibodies in sera from hyperimmune Saimiri monkeys. Using Thiazole Orange dye for detection of parasitised cells, this analysis was performed on a FACSscan apparatus equipped with a single laser.

Adult↗
PubMed1994

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

Animals↗
PubMed1994

Immunogenicity and antigenicity of a Plasmodium falciparum protein fraction (90-110 kDa) able to protect squirrel monkeys against asexual blood stages.

A monkey vaccination trial using a Plasmodium falciparum protein fraction containing antigens of 90-110 kDa is reported. The fraction was obtained by electroelution from preparative polyacrylamide gels. Three monkeys out of five resisted a heavy challenge dose of highly virulent parasites. Using specific antisera, several components of the fraction were identified, namely heat shock protein 90 (hsp90), Ag44/RhopH3, ABRA, 96tR/GBP130 and Pf96 protease. The fraction did not contain KAHRP, nor the SERP antigen. The antibody response of the monkeys was studied on these individual antigens purified by preparative immunoprecipitation. Surprisingly, hsp90 was found in the immunoprecipitates obtained with SERP antisera. Interestingly, the response to hsp90 correlated with protection, high antibody titres being found only in the protected monkeys. In contrast, no correlation with protection could be found for the response to the other antigens.

Animals↗
PubMed1993

In vitro phagocytosis inhibition assay for the screening of potential candidate antigens for sub-unit vaccines against the asexual blood stage of Plasmodium falciparum.

We have previously established a direct correlation between immune protection against the asexual blood stage Plasmodium falciparum infection and the presence of opsonizing antibodies promoting phagocytosis of parasitized red blood cells. In the present communication we describe an in vitro assay for measuring phagocytosis inhibition (PIA) specific for P. falciparum-infected erythrocytes. The phagocytosis inhibition assay is a simple procedure for screening potential candidates for sub-unit vaccines against P. falciparum based on the correlation between opsonizing antibodies and immunoprotection. The assay was used to analyse 18 recombinant molecules, corresponding to 11 distinct antigens of P. falciparum. Pre-incubation and selective antibody depletion experiments demonstrate the antigen-antibody specificity of the PIA. The presence of epitopes participating as targets of opsonic antibodies were demonstrated in six distinct polypeptide antigens.

Animals↗
PubMed1992

Gene inactivation of Pf11-1 of Plasmodium falciparum by chromosome breakage and healing: identification of a gametocyte-specific protein with a potential role in gametogenesis.

We report the identification of the product of the Plasmodium falciparum Pf11-1 gene and demonstrate that it is a gametocyte-specific protein that has a potential role in the rupture of the host erythrocyte and emergence of the gametes (gametogenesis). The Pf11-1 gene is a large locus (30 kb) whose sequence predicts a glutamic acid-rich polypeptide. Our identification of the Pf11-1 gene product as gametocyte specific was greatly facilitated by the isolation of a mutant parasite clone in which greater than 90% of the Pf11-1 gene was deleted. Molecular analysis of the mutant locus suggests that the underlying genetic mechanism is chromosome breakage and subsequent healing by the addition of telomere repeats. PCR-based analysis showed that similar DNA rearrangements occur commonly in small subpopulations of most laboratory strains, suggesting that the Pf11-1 locus represents a fragile chromosome region. Northern blot analysis demonstrates that a large Pf11-1 gene-specific transcript (much greater than 10 kb) is present in gametocytes but not in asexual blood stage parasites. The Pf11-1 protein was localized by electron microscopy to granules in the cytoplasm of gametocytes adjacent to the membrane of the parasitophorous vacuole. Following in vitro stimulation of gametogenesis, the Pf11-1 protein was found in the membrane of lysed erythrocytes, suggesting a role for Pf11-1 in erythrocyte rupture within the mosquito gut.

Animals↗
PubMed1992

Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities.

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.

Animals↗
PubMed1990

Genetic aspects of malaria parasite infection and the host immune response in relation to parasite evasion.

In relation to problems found up to now for the development of vaccines against malaria, recent studies have pointed out the different strategies evolved by the parasites to evade the host immune response. They include antigenic variation, antigenic polymorphism, antigenic mimicry and direct interference with the generation of the host immune response. These different mechanisms are rapidly reviewed and discussed with the aim of focusing the actual orientation of basic and applied research which has been developed to overcome these difficulties.

Animals↗
PubMed1989

Acute virulent infection with Plasmodium chabaudi does not impair the generation of a protective immune response.

We have investigated whether a protective immune response occurred in mice infected with a virulent cloned strain of Plasmodium chabaudi. Animals inoculated intravenously with 10(7) parasitized erythrocytes (PE) showed an exponentially increasing parasitaemia and died by day 6 of the infection, presenting a pronounced anaemia. Smaller inocula produced a longer pre-patent period but did not change the lethal course of infection, since mice injected with 100 parasites died on day 12. When anaemia was compensated for by red blood cell (RBC) transfusion, infected mice recovered and thereafter exhibited a strong immunity, comparable to that of mice immunized by a drug-controlled infection. The immune response was P. chabaudi specific, as the mice were fully susceptible to a challenge by P. yoelii. Three transfusions of 5 x 10(9) RBC per mouse at 2-day intervals were necessary before all the animals were able to control the infection. Transfusion of a larger number of RBC resulted in a lower anaemia and a delay in reticulocytaemia but, paradoxically, the expression of the immune response was delayed. Three transfusions of 1.2 x 10(10) RBC enabled three out of eight mice to survive the infection, while six transfusions enabled all the mice to survive. The data suggest that parasitized immature RBC could play an important role in triggering the protective immune response.

Animals↗
PubMed1988

Activation of a Plasmodium falciparum protease correlated with merozoite maturation and erythrocyte invasion.

Protease-dependent processes of the P. falciparum schizogonic cycle are briefly described. The P. falciparum p76 protease is the first example of a biochemically regulated protease, the activation of which is related to merozoite maturation and/or erythrocyte invasion. The main known properties of the p76 protease are reviewed and some original results concerning its biosynthesis and biological properties are described.

Animals↗
PubMed1987

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin.

Yeast submitochondrial particles, in a Pi- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5-10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.

Adenosine Triphosphate↗
PubMed1987

Isotypic pattern of the polyclonal B cell response during primary infection by Plasmodium chabaudi and in immune-protected mice.

The primary infection by P. chabaudi induces an increase of the numbers of splenic immunoglobulin (Ig)-secreting B cells in both athymic and euthymic BALB/c mice. The isotypic pattern of the polyclonal response is restricted only in euthymic mice where IgG2a, IgG2b and IgM plaque-forming cells (PFC) predominate. In immunized animals, protected against a parasite challenge, the isotypic pattern of splenic PFC is completely different, the IgG1 and IgM isotypes constituting the main part of the response. Reinoculation of immune-protected animals induces a PFC response which is dose dependent and accentuates the characteristic isotypic profile of the immune-protected mice.

Animals↗
PubMed1987

The polymorphic 11.1 locus of Plasmodium falciparum.

Clone pPF11.1 encodes a Plasmodium falciparum antigen expressed during the intraerythrocytic cycle and containing tandem repeats of a 9 amino acid unit. We report here an analysis of the genomic region specific for 11.1, which extends over 30 kb. It contains two blocks of repeats, spanning 13 kb and 9 kb. The restriction map suggests that the locus may result from a gene duplication. The 11.1 region is present in all P. falciparum strains examined so far. Southern analysis of 8 distinct isolates indicates that the locus is highly polymorphic. Thus the pPF11.1 repeats constitute a sensitive and discriminating probe to type P. falciparum strains.

Alleles↗
PubMed1987

Polypeptide antigens Mr 90,000 and 72,000 related to protective immunity against the blood form of Plasmodium falciparum in the squirrel monkey show stable characteristics in strains from different geographic origins.

Squirrel monkeys protected after vaccination with a particular protein fraction of Plasmodium falciparum elicit antibodies directed against two parasite proteins, Mr 90,000 and 72,000. We have used monoclonal antibodies and sera from protected monkeys to determine whether or not these polypeptides were polymorphic in 22 strains. In all the isolates studied, both polypeptides were conserved, as was the epitope defined by monoclonal antibody XIV/7 present on the Mr 90,000. Immunofluorescence of all strains showed the same pattern using 5 Mab produced against different fragments of the Mr 72,000 polypeptide from the FUP strain. All isolates examined were positive, indicating that this polypeptide was present in different strains and that the 5 epitopes were conserved. Peptide mapping of both the Mr 90,000 and 72,000 antigen purified from 3 different strains indicated that each antigen appeared to be conserved.

Africa↗
PubMed1986

t-Butylhydroperoxide-induced Ca2+ efflux from liver mitochondria in the presence of physiological concentrations of Mg2+ and ATP.

Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM ATP, 4 mM Mg2+ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca2+, a small decrease in delta psi and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca2+ cycling was prevented by ruthenium red, the changes in delta psi, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca2+ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides.

Acetoacetates↗