Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP1(19), PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP1(19). Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines.

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

Dietary proteins and atherosclerosis.

More than one hundred years ago the "protein hypothesis" of the pathogenesis of atherosclerosis and its association with cardiovascular disease was put forward on the basis of animal experiments; however, it has so far never been verified in humans. This theory was soon replaced by the "lipid hypothesis", which was confirmed in humans as of 1994. Epidemiological ecological studies in the 1960 s showed significant associations between dietary animal protein and mortality from cardiovascular disease. However, animal protein intake was also significantly correlated with saturated fatty acid and cholesterol intake. In the last decades two prospective cohort studies demonstrated a decreased cardiovascular risk in women during high- versus low-protein intake when adjusting for other dietary factors (e. g., saturated fats) and other cardiovascular risk factors. A direct cholesterol lowering effect of proteins has not been shown. Despite earlier research indicating that soy protein has cardioprotective effects as compared to other proteins, these observations have not been confirmed by randomized placebo-controlled trials. However, most experts recommend the consumption of foods rich in plant proteins as alternatives to meat and dairy products rich in saturated fat and containing cholesterol. There are no scientific arguments to increase the daily protein intake to more than 20 % of total energy intake as recommended by the guidelines, in order to improve cardiovascular health.

Pathogenesis-related proteins and the jasmonate signaling pathway

Résumé Etant une importante source d'énergie, les plantes sont constamment attaquées par des pathogènes. Ne pouvant se mouvoir, elles ont développé des systèmes de défense sophistiqués afin de lutter contre ces prédateurs. Parmi ces systèmes, les voies de signalisation mettant en jeu des éliciteurs endog8nes tels que les jasmonates permettent d'induire la production de protéines de défense telles que les protéines dites "liées à la pathogénèse". Les gènes codant pour ces protéines appartiennent à des familles multigéniques. Le premier but de cette thèse est d'évaluer le nombre de ces gènes dans le génome d'Arabidopsis thaliana et d'estimer la part de ce système de défense, dépendant de la voie de signalisation des jasmonates. Nous avons défini un cluster de seulement 1S gènes sur 266, "liés à la pathogénèse", exclusivement régulés par les jasmonates. De multiples membres des familles des lectines de type jacaline et des inhibiteurs de trypsines semblent dépendre du jasmonate. Présente dans tous les systèmes immunitaires des eucaryotes, la famille des défensines est une famille très intéressante. Chez Arabidopsis thaliana, 317 protéines similaires aux défensines ont été définies, cependant seulement 15 défensines (PDF) sont bien annotées. Ces 15 défensines sont séparées en deux groupes dont un semble avoir évolué plus récemment. Le second but de cette thèse est d'étudier ce groupe de défensines à l'aide de la bioinformatique et des techniques de biologie moléculaire (gêne rapporteur, PCR en temps réel). Nous avons montré que ce groupe contenait une défensine acide intéressante, PDF1.5, qui semblait avoir subi une sélection positive. Cette protéine n'avait encore jamais été étudiée. Contrairement à ce que nous pensions, nous avons établi que cette protéine pouvait avoir une activité biologique liée à la défense. Ce travail de thèse a permis de préciser le nombre de gènes "liées à la pathogénèse" induits par la voie des jasmonates et d'apporter des éléments de réponse sur la question de la redondance des gènes de défense. En conclusion, même si de nombreuses familles de gènes intervenant dans la défense sont bien définies chez Arabidopsis, il reste encore de nombreuses études à faire sur chacun de ces membres. Abstract Being an important source of energy, plants are constantly attacked by herbivores and pathogens. As sessile organisms, they have developed sophisticated defense responses to cope with attack. Among these responses, signalling pathways, using endogenous elicitors including jasmonates (JA), allow the plant to induce the production of defense proteins such as pathogenesis-related (PR) proteins. The genes encoding these proteins belong to multigenic families. The first goal of this thesis was to evaluate the number of PR genes in the genome of Arabidopsis thaliana and estimate how much of this plant defense system was dependent on the jasmonate signaling pathway in leaves. Surprisingly a cluster of only 1S genes out of 2ó6 PR genes was exclusively regulated by JA. Multiple members of the jacalin lectin and trypsin inhibitor gene families were shown to be regulated by JA. Present in all eukaryotic immune systems, defensins are an attractive PR family to study. In Arabidopsis thaliana, 317 defensin-related proteins have been found but just 1S defensins (i.e. PDF family) are well annotated. These defensins are split into 2 groups. One of these groups may have appeared and diversified recently. The second goal of this thesis was to study this defensin gene group combining bioinformatic, reporter gene and quantitative PCR techniques. We have shown that this group contains an interesting acidic defensin, PDF1.S, which seems to have undergone positive selection. No information was known on this protein. We have established that this protein may have a biological activity in plant defense. This thesis allowed us to define the number of PR genes induced by the jasmonate pathway and gave initial leads to explain the redundancy of the PR genes in the genome of Arabidopsis. In conclusion, even if many defense gene families are already defined in the Arabidopsis genome, much work remains to be done on individual members.

Serosurvey Study of Toscana Virus in Domestic Animals, Granada, Spain

Toscana virus (TOSV) is transmitted by infected sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis in humans. It remains unclear if there are animal reservoirs able to maintain the virus through the cold months of the year, when the vector is not circulating. From May to October of 2006 and 2007, we conducted a serosurvey study on domestic animals from Granada province (southern Spain). TOSV was investigated in 1186 serum samples from horses, goats, pigs, cats, dogs, sheep, and cows by serology (indirect fluorescence assay), viral culture, and RT-polymerase chain reaction. Specific anti-TOSV antibodies were detected in 429 (36.2%) serum samples. The highest seropositivity rates were observed in cats (59.6%) and dogs (48.3%). These results suggest that an important percentage of the domestic animals have been infected by TOSV. Significantly different seroprevalence rates were detected in goats among distinct geographical areas. All viral cultures were negative. TOSV was detected by RT-polymerase chain reaction in only one serum sample from a goat. Thus, the studied animals do not seem to act as reservoirs for TOSV; otherwise, they could be amplifying hosts for the virus.

Influence of HLA DRB1 alleles in the susceptibility of rheumatoid arthritis and the regulation of antibodies against citrullinated proteins and rheumatoid factor.

INTRODUCTION The purpose of this study was to investigate the association between HLA-DRB1 alleles with susceptibility to rheumatoid arthritis (RA) and production of antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF). METHODS We studied 408 patients (235 with RA, 173 non-RA) and 269 controls. ACPA, RF and HLA-DR typing were determined. RESULTS We found an increased frequency of HLA DRB1 alleles with the shared epitope (SE) in ACPA-positive RA. Inversely, HLA DRB1 alleles encoding DERAA sequences were more frequent in controls than in ACPA-positive RA, and a similar trend was found for HLA DR3. However, these results could not be confirmed after stratification for the presence of the SE, probably due to the relatively low number of patients. These data may suggest that the presence of these alleles may confer a protective role for ACPA-positive RA. In RA patients we observed association between SE alleles and ACPA titers in a dose-dependent effect. The presence of HLA DR3 or DERAA-encoding alleles was associated with markedly reduced ACPA levels. No association between RF titers and HLA DR3 or DERAA-encoding alleles was found. CONCLUSIONS HLA DRB1 alleles with the SE are associated with production of ACPA. DERAA-encoding HLA-DR alleles and HLA DR3 may be protective for ACPA-positive RA.

Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4-diacetylphloroglucinol really synonymous with higher plant protection?

The antifungal compound 2,4-diacetylphloroglucinol (Phl) contributes to biocontrol in pseudomonads, but whether or not Phl(+) biocontrol pseudomonads display higher plant-protecting activity than Phl(-) biocontrol pseudomonads remains to be demonstrated. This issue was addressed by assessing 230 biocontrol fluorescent pseudomonads selected from a collection of 3132 bacterial isolates obtained from 63 soils worldwide. One-third of the biocontrol pseudomonads were Phl(+) and almost all Phl(+) isolates also produced hydrogen cyanide (HCN). The only Phl(+) HCN(-) strain did harbor hcn genes, but with the deletion of a 134 bp hcnC fragment corresponding to an ADP-binding motif. Statistical analysis of biocontrol isolate distributions indicated that Phl production ability was associated with superior disease suppression activity in the Pythium-cucumber and Fusarium-tomato pathosystems, but this was also the case with HCN production ability. However, HCN significance was not as strong, as indicated both by the comparison of Phl(-) HCN(+) and Phl(-) HCN(-) strains and by correlation analyses. This is the first population-level demonstration of the higher plant-protecting activity of Phl(+) biocontrol pseudomonads in comparison with Phl(-) biocontrol pseudomonads.

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation - A Review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.

Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development.

The cytoskeleton is important for neuronal morphogenesis. During the postnatal development of cat brain, the molecular composition of the neuronal cytoskeleton changes with maturation. Several of its proteins change in their rate of expression, in their degree of phosphorylation, in their subcellular distribution, or in their biochemical properties. It is proposed that phosphorylation is an essential mechanism to regulate the plasticity of the early, juvenile-type cytoskeleton. Among such proteins are several microtubule-associated proteins (MAPs), such as MAP5a, MAP2c or the juvenile tau proteins. Phosphorylation may also act on neurofilaments, postulated to be involved in the adult-type stabilization of axons. These observations imply that phosphorylation may affect cytoskeleton function in axons and dendrites at various developmental stages. Yet, the mechanisms of phosphorylation and its regulation cascades are largely unknown. In view of the topic of this issue on CD15, the potential role of matrix molecules being involved in the modulation of phosphorylation activity and of cytoskeletal properties is addressed.

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease.

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development.

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton.