Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36.

Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of ß-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and ß-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where ß-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.

Integrative Control Between Proton Pumps and SOS1 Antiporters in Roots is Crucial for Maintaining Low Na+ Accumulation and Salt Tolerance in Ammonium-Supplied Sorghum bicolor.

An effective strategy for re-establishing K+ and Na+ homeostasis is a challenge for the improvement of plant performance in saline soil. Specifically, attempts to understand the mechanisms of Na+ extrusion from plant cells, the control of Na+ loading in the xylem and the partitioning of the accumulated Na+ between different plant organs are ongoing. Our goal was to provide insight into how an external nitrogen source affects Na+ accumulation in Sorghum bicolor under saline conditions. The NH4+ supply improved the salt tolerance of the plant by restricting Na+ accumulation and improving the K+/Na+ homeostasis in shoots, which was consistent with the high activity and expression of Na+/H+ antiporters and proton pumps in the plasma membrane and vacuoles in the roots, resulting in low Na+ loading in the xylem. Conversely, although NO3--grown plants had exclusion and sequestration mechanisms for Na+, these responses were not sufficient to reduce Na+ accumulation. In conclusion, NH4+ acts as an efficient signal to activate co-ordinately responses involved in the regulation of Na+ homeostasis in sorghum plants under salt stress, which leads to salt tolerance.

Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Structural basis for NKG2A/CD94 recognition of HLA-E.

The NKG2x/CD94 (x = A, C, E) natural killer-cell receptors perform an important role in immunosurveillance by binding to HLA-E complexes that exclusively present peptides derived from MHC class I leader sequences, thereby monitoring MHC class I expression. We have determined the crystal structure of the NKG2A/CD94/HLA-E complex at 4.4-A resolution, revealing two critical aspects of this interaction. First, the C-terminal region of the peptide, which displays the most variability among class I leader sequences, interacts entirely with CD94, the invariant component of these receptors. Second, residues 167-170 of NKG2A/C account for the approximately 6-fold-higher affinity of the inhibitory NKG2A/CD94 receptor compared to its activating NKG2C/CD94 counterpart. These residues do not contact HLA-E or peptide directly but instead form part of the heterodimer interface with CD94. An evolutionary analysis across primates reveals that whereas CD94 is evolving under purifying selection, both NKG2A and NKG2C are evolving under positive selection. Specifically, residues at the CD94 interface have evolved under positive selection, suggesting that the evolution of these genes is driven by an interaction with pathogen-derived ligands. Consistent with this possibility, we show that NKG2C/CD94, but not NKG2A/CD94, weakly but specifically binds to the CMV MHC-homologue UL18. Thus, the evolution of the NKG2x/CD94 family of receptors has likely been shaped both by the need to bind the invariant HLA-E ligand and the need to avoid subversion by pathogen-derived decoys.

A method for the identification of guinea pig blood meal in the Chagas disease vector, Triatoma infestans.

BACKGROUND: In a SINE-based PCR assay, a primer set specific for guinea pig genome short interspersed elements DNA was used to test the utility of genomic markers for identifying the source of vertebrate blood meals of Triatoma infestans. METHODS: The investigation consisted of two assays. In Assay 1, thirty-six insects, collected from the Province of Zudáñez in Chuquisaca, Bolivia were frozen 1-40 hours after feeding, under controlled conditions, on guinea pigs. The species of the vertebrate host was confirmed from dissection of the posterior part of the abdomen of each insect followed by DNA extraction and PCR amplification. Assay 2 investigated whether the technique worked under field conditions. We analyzed the bloodmeal of 34 insects collected from households and peri-domestic structures from communities where wild and captive guinea pigs occur. After collection, the insects were maintained at room temperature for 2 months without feeding and then analyzed. RESULTS: In Assay 1, each of the 36 insects allowed to feed on guinea pig blood tested positive for guinea pig DNA. The guinea pig DNA was reliably identified in as little as 1 hour and up to 40 hours after feeding. For Assay 2, 8 out of the 34 samples (23%) showed positive results with guinea pig specific primers. CONCLUSION: The results in assay 1 demonstrated that DNA from the vertebrate host can be amplified 1-40 hours post feeding from the abdomen of the blood-feeding Chagas disease vector Triatoma infestans. The results in assay 2 confirmed that the procedure works on insects collected from households and peri-domestic structures and that the source of a blood meal can be determined at least 2 months post feeding.

PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: high rates found in Chuquisaca, Bolivia.

BACKGROUND: The Andean valleys of Bolivia are the only reported location of sylvatic Triatoma infestans, the main vector of Chagas disease in this country, and the high human prevalence of Trypanosoma cruzi infection in this region is hypothesized to result from the ability of vectors to persist in domestic, peri-domestic, and sylvatic environments. Determination of the rate of Trypanosoma infection in its triatomine vectors is an important element in programs directed at reducing human infections. Traditionally, T. cruzi has been detected in insect vectors by direct microscopic examination of extruded feces, or dissection and analysis of the entire bug. Although this technique has proven to be useful, several drawbacks related to its sensitivity especially in the case of small instars and applicability to large numbers of insects and dead specimens have motivated researchers to look for a molecular assay based on the polymerase chain reaction (PCR) as an alternative for parasitic detection of T. cruzi infection in vectors. In the work presented here, we have compared a PCR assay and direct microscopic observation for diagnosis of T. cruzi infection in T. infestans collected in the field from five localities and four habitats in Chuquisaca, Bolivia. The efficacy of the methods was compared across nymphal stages, localities and habitats. METHODS: We examined 152 nymph and adult T. infestans collected from rural areas in the department of Chuquisaca, Bolivia. For microscopic observation, a few drops of rectal content obtained by abdominal extrusion were diluted with saline solution and compressed between a slide and a cover slip. The presence of motile parasites in 50 microscopic fields was registered using 400x magnification. For the molecular analysis, dissection of the posterior part of the abdomen of each insect followed by DNA extraction and PCR amplification was performed using the TCZ1 (5' - CGA GCT CTT GCC CAC ACG GGT GCT - 3') and TCZ2 (5' - CCT CCA AGC AGC GGA TAG TTC AGG - 3') primers. Amplicons were chromatographed on a 2% agarose gel with a 100 bp size standard, stained with ethidium bromide and viewed with UV fluorescence. For both the microscopy and PCR assays, we calculated sensitivity (number of positives by a method divided by the number of positives by either method) and discrepancy (one method was negative and the other was positive) at the locality, life stage and habitat level. The degree of agreement between PCR and microscopy was determined by calculating Kappa (k) values with 95% confidence intervals. RESULTS: We observed a high prevalence of T. cruzi infection in T. infestans (81.16% by PCR and 56.52% by microscopy) and discovered that PCR is significantly more sensitive than microscopic observation. The overall degree of agreement between the two methods was moderate (Kappa = 0.43 +/- 0.07). The level of infection is significantly different among communities; however, prevalence was similar among habitats and life stages. CONCLUSION: PCR was significantly more sensitive than microscopy in all habitats, developmental stages and localities in Chuquisaca, Bolivia. Overall we observed a high prevalence of T. cruzi infection in T. infestans in this area of Bolivia; however, microscopy underestimated infection at all levels examined.

Putative role of glutamine in the activation of CBL/CIPK signalling pathways during salt stress in sorghum.

The salt overly sensitive (SOS) pathway is the only mechanism known for Na+ extrusion in plant cells. SOS pathway activation involves Ca2+-sensing proteins, such as calcineurin B-like (CBL) proteins, and CBL-interacting protein kinases (CIPKs). In this signalling mechanism, a transit increase in cytosolic Ca2+ concentration triggered by Na+ accumulation is perceived by CBL (also known as SOS3). Afterward, SOS3 physically interacts with a CIPK (also known as SOS2), forming the SOS2/SOS3 complex, which can regulate the number downstream targets, controlling ionic homeostasis. For instance, the SOS2/SOS3 complex phosphorylates and activates the SOS1 plasmalemma protein, which is a Na+/H+ antiporter that extrudes Na+ out of the cell. The CBL-CIPK networking system displays specificity, complexity and diversity, constituting a critical response against salt stress and other abiotic stresses. In a study reported in the journal Plant and Cell Physiology, we showed that NH4+ induces the robust activation of transporters for Na+ homeostasis in root cells, especially the SOS1 antiporter and plasma membrane H+-ATPase, differently than does NO3-. Despite some studies having shown that external NH4+ ameliorates salt-induced effects on ionic homeostasis, there is no evidence that NH4+ per se or some product of its assimilation is responsible for these responses. Here, we speculate about the signalling role behind glutamine in CBL-CIPK modulation, which could effectively activate the SOS pathway in NH4+-fed stressed plants.

Structural comparison of apical membrane antigen 1 orthologues and paralogues in apicomplexan parasites.

Apical membrane antigen 1 (AMA1) is a membrane protein present in Plasmodium species and is probably common to all apicomplexan parasites. The recent crystal structure of the complete ectoplasmic region of AMA1 from Plasmodium vivax has shown that it comprises three structural domains and that the first two domains are based on the PAN folding motif. Here, we discuss the consequences of this analysis for the three-dimensional structure of AMA1 from other Plasmodium species and other apicomplexan parasites, and for the Plasmodium paralogue MAEBL. Many polar and apolar interactions observed in the PvAMA1 crystal structure are made by residues that are invariant or highly conserved throughout all Plasmodium orthologues; a subgroup of these residues is also present in other apicomplexan orthologues and in MAEBL. These interactions presumably play a key role in defining the protein fold. Previous studies have shown that the ectoplasmic region of AMA1 must be cleaved from the parasite surface for host-cell invasion to proceed. The cleavage site in the crystal structure is not readily accessible to proteases and we discuss possible consequences of this observation. The three-dimensional distribution of polymorphic sites in PfAMA1 shows that these are all on the surface and that their positions are significantly biased to one side of the ectoplasmic region. Of particular note, a flexible segment in domain II, comprising about 40 residues and devoid of polymorphism, carries an epitope recognized by an invasion-inhibitory monoclonal antibody and a T-cell epitope implicated in the human immune response to AMA1.

Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

BACKGROUND: In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. METHODOLOGY/PRINCIPAL FINDINGS: We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). CONCLUSIONS/SIGNIFICANCE: We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

Exploring the Trypanosoma brucei Hsp83 potential as a target for structure guided drug design.

Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs--whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases. In this paper, we report the exploration of T. brucei Hsp83--a homolog of human Hsp90--as a drug target using multiple biophysical and biochemical techniques. Our approach included the characterization of the chemical sensitivity of the parasitic chaperone against a library of known Hsp90 inhibitors by means of differential scanning fluorimetry (DSF). Several compounds identified by this screening procedure were further studied using isothermal titration calorimetry (ITC) and X-ray crystallography, as well as tested in parasite growth inhibitions assays. These experiments led us to the identification of a benzamide derivative compound capable of interacting with TbHsp83 more strongly than with its human homologs and structural rationalization of this selectivity. The results highlight the opportunities created by subtle structural differences to develop new series of compounds to selectively target the Trypanosoma brucei chaperone and effectively kill the sleeping sickness parasite.

Crystal structure of the complex mAb 17.2 and the C-terminal region of Trypanosoma cruzi P2ß protein: implications in cross-reactivity.

Patients with Chronic Chagas' Heart Disease possess high levels of antibodies against the carboxyl-terminal end of the ribosomal P2ß protein of Trypanosoma cruzi (TcP2ß). These antibodies, as well as the murine monoclonal antibody (mAb) 17.2, recognize the last 13 amino acids of TcP2ß (called the R13 epitope: EEEDDDMGFGLFD) and are able to cross-react with, and stimulate, the ß1 adrenergic receptor (ß1-AR). Indeed, the mAb 17.2 was able to specifically detect human ß1-AR, stably transfected into HEK cells, by flow cytometry and to induce repolarisation abnormalities and first degree atrioventricular conduction block after passive transfer to naïve mice. To study the structural basis of this cross-reactivity, we determined the crystal structure of the Fab region of the mAb 17.2 alone at 2.31 Å resolution and in complex with the R13 peptide at 1.89 Å resolution. We identified as key contact residues on R13 peptide Glu3, Asp6 and Phe9 as was previously shown by alanine scanning. Additionally, we generated a model of human ß1-AR to elucidate the interaction with anti-R13 antibodies. These data provide an understanding of the molecular basis of cross-reactive antibodies induced by chronic infection with Trypanosoma cruzi.

NH(4)(+)-stimulated low-K(+) uptake is associated with the induction of H(+) extrusion by the plasma membrane H(+)-ATPase in sorghum roots under K(+) deficiency.

The effect of external inorganic nitrogen and K(+) content on K(+) uptake from low-K(+) solutions and plasma membrane (PM) H(+)-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4mM K(+) and inorganic nitrogen as NO(3)(-), NO(3)(-)/NH(4)(+) or NH(4)(+) and then starved of K(+) for 24, 48 and 72 h. NH(4)(+) in full nutrient solution significantly affected the uptake efficiency and accumulation of K(+), and this effect was less pronounced at the high K(+) concentration. In contrast, the translocation rate of K(+) to the shoot was not altered. Depletion assays showed that plants grown with NH(4)(+) more efficiently depleted the external K(+) and reached higher initial rates of low-K(+) uptake than plants grown with NO(3)(-). One possible influence of K(+) content of shoot, but not of roots, on K(+) uptake was evidenced. Enhanced K(+)-uptake capacity was correlated with the induction of H(+) extrusion by PM H(+)-ATPase. In plants grown in high K(+) solutions, the increase in the active H(+) gradient was associated with an increase of the PM H(+)-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2mM K(+), only the initial rate of H(+)-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H(+)-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO(3)(-)-grown plants. The results suggest that K(+) homeostasis in NH(4)(+)-grown sorghum plants may be regulated by a high capacity for K(+) uptake, which is dependent upon the H(+)-pumping activity of PM H(+)-ATPase.

A new method for forensic DNA analysis of the blood meal in chagas disease vectors demonstrated using Triatoma infestans from Chuquisaca, Bolivia.

BACKGROUND: Feeding patterns of the vector are important in the epidemiology of Chagas disease, the leading cause of heart disease in Latin America. Chagas disease is caused by the parasite, Trypanasoma cruzi, which is transmitted by blood feeding insects. Historically, feeding behaviours of haematophagous insects have been investigated using serological reactions, which have detection limits in terms of both taxonomic resolution, and quantity and quality of the blood meal. They are labor intensive, require technical expertise, need fresh or frozen samples and antibodies often are either not available commercially or the resources for synthesis and purification are not available. We describe an assay to identify vertebrate blood meal sources, and the parasite T. cruzi using species-specific PCR assays from insect vectors and use the method to provide information regarding three questions: (1) Do domestic and peri-domestic (chicken coop and animal corral) habitats vary in the blood meals detected in the vectors? (2) What is the pattern of multiple blood meals? (3) Does the rate of T. cruzi infection vary among habitats and is it associated with specific blood meal types? METHODOLOGY/PRINCIPAL FINDINGS: Assays based on the polymerase chain reaction were evaluated for identification of the blood meal source in the heamatophagous Chagas disease vector Triatoma infestans. We evaluate a technique to identify 11 potential vertebrate food sources from the complex mixture extracted from the vector's abdomen. We tested the assay on 81 T. infestans specimens collected from the Andean highlands in the department of Chuquisaca, located in central Bolivia, one of the regions in South America where sylvatic T. infestans have been reported. This area is suggested to be the geographic origin of T. infestans and has very high human infection rates that may be related to sylvatic vector populations. CONCLUSION/SIGNIFICANCE: The results of the assays revealed that a high percentage of insects collected in human dwellings had fed on peri-domestic animals. In contrast, one insect from a chicken coop but no bugs from corrals tested positive for human blood. Forty-eight percent of insects tested positive for more than one vertebrate species. T. cruzi infection was detected in 42% of the specimens. From the epidemiological point of view, the results reveal an overall pattern of movement from peri-domestic structures to human habitations for T. infestans in this region of Bolivia as well as the important role of pigs, dogs, chickens and guinea pigs in the dynamics of T. cruzi infection.

Microsatellites reveal a high population structure in Triatoma infestans from Chuquisaca, Bolivia.

BACKGROUND: For Chagas disease, the most serious infectious disease in the Americas, effective disease control depends on elimination of vectors through spraying with insecticides. Molecular genetic research can help vector control programs by identifying and characterizing vector populations and then developing effective intervention strategies. METHODS AND FINDINGS: The population genetic structure of Triatoma infestans (Hemiptera: Reduviidae), the main vector of Chagas disease in Bolivia, was investigated using a hierarchical sampling strategy. A total of 230 adults and nymphs from 23 localities throughout the department of Chuquisaca in Southern Bolivia were analyzed at ten microsatellite loci. Population structure, estimated using analysis of molecular variance (AMOVA) to estimate F(ST) (infinite alleles model) and R(ST) (stepwise mutation model), was significant between western and eastern regions within Chuquisaca and between insects collected in domestic and peri-domestic habitats. Genetic differentiation at three different hierarchical geographic levels was significant, even in the case of adjacent households within a single locality (R(ST) = 0.14, F(ST) = 0.07). On the largest geographic scale, among five communities up to 100 km apart, R(ST) = 0.12 and F(ST) = 0.06. Cluster analysis combined with assignment tests identified five clusters within the five communities. CONCLUSIONS: Some houses are colonized by insects from several genetic clusters after spraying, whereas other households are colonized predominately by insects from a single cluster. Significant population structure, measured by both R(ST) and F(ST), supports the hypothesis of poor dispersal ability and/or reduced migration of T. infestans. The high degree of genetic structure at small geographic scales, inferences from cluster analysis and assignment tests, and demographic data suggest reinfesting vectors are coming from nearby and from recrudescence (hatching of eggs that were laid before insecticide spraying). Suggestions for using these results in vector control strategies are made.

Crystal structure of the tryparedoxin peroxidase from the human parasite Trypanosoma cruzi.

Tryparedoxin peroxidase from Trypanosoma cruzi (TcTXNPx) belongs to the family of typical 2-Cys peroxiredoxins. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. In T. cruzi, as in all trypanosomatids, this enzyme is the final electron acceptor of a unique system for detoxifying hydroperoxides, constituting a relevant target for drug design. We have determined the crystal structure of TcTXPNx in the reduced active state. The structure comprises 10 subunits in the asymmetric unit, associated to form a decamer of toroidal shape obeying 52 (D5) point group symmetry. We have analyzed the structure of TcTXNPx by comparing it with other structures of typical 2-Cys peroxiredoxins in both redox states, and have identified key residues in the structural rearrangement taking place in the enzymatic cycle. This is the first report of the structure of an active peroxiredoxin that has peroxidase and peroxynitrite reductase activity, and it is noteworthy that it is from a human parasite. This knowledge is of interest for further understanding peroxide metabolism in these parasites, and in the design of new trypanosomatidal drugs against Chagas disease.

Crystal structure of the malaria vaccine candidate apical membrane antigen 1.

Apical membrane antigen 1 from Plasmodium is a leading malaria vaccine candidate. The protein is essential for host-cell invasion, but its molecular function is unknown. The crystal structure of the three domains comprising the ectoplasmic region of the antigen from P. vivax, solved at 1.8 angstrom resolution, shows that domains I and II belong to the PAN motif, which defines a superfamily of protein folds implicated in receptor binding. We also mapped the epitope of an invasion-inhibitory monoclonal antibody specific for the P. falciparum ortholog and modeled this to the structure. The location of the epitope and current knowledge on structure-function correlations for PAN domains together suggest a receptor-binding role during invasion in which domain II plays a critical part. These results are likely to aid vaccine and drug design.

Somatic mutations can lead to a loss of superantigenic and polyreactive binding.

Although antibodies have been assumed to bind a specific antigen, evidence exists showing that a single antibody can bind to multiple unrelated antigens. We previously studied a human monoclonal antibody expressing a mutated form of the V(H)3-73 gene and displaying anti-tubulin activity in a patient suffering from an immunocytic lymphoma. Despite its expression of a V(H)3 family member, this immunoglobulin failed to react with protein A (SpA), suggesting that somatic mutations could account for its change in specificity. To examine this possibility, we produced recombinant Ig expressing germ-line (IgM kappa-Germ) or the mutated form (IgM kappa-PER) of the V(H)3-73 fragment. Comparison of the respective affinities of the two Ig demonstrated that IgM kappa-Germ restores its SpA-binding capacity, and shows a moderate decrease in its affinity for tubulin. Interestingly, IgM kappa-Germ displayed polyreactive specificity for different autoantigens, which contrasted to the monospecific binding of IgM kappa-PER to tubulin. These results suggest that the monoreactive IgM kappa-PER antibody may be derived from a natural polyreactive antibody through somatic mutation. In addition, both temperature modification and mild denaturation succeeded in recovering the polyreactivity of IgM kappa-PER, which favors the view that conformational modifications of the tertiary structure of antibodies may play a key role in the genesis of polyreactivity.

Expression, crystallization and preliminary structural analysis of the ectoplasmic region of apical membrane antigen 1 from Plasmodium vivax, a malaria-vaccine candidate.

Apical membrane antigen 1 (AMA1), a type 1 transmembrane protein present in the microneme organelles of Plasmodium, is a leading malaria-vaccine candidate. The ectoplasmic region of AMA1 from P. vivax has been expressed in Pichia pastoris and crystallized in two different forms: an orthorhombic form (space group P2(1)2(1)2(1), unit-cell parameters a = 54.1, b = 76.1, c = 103.9 A) and a monoclinic form (space group C2, unit-cell parameters a = 150.0, b = 53.8, c = 60.3 A, beta = 113.2 degrees ). Native data have been collected to 2.0 A resolution for the orthorhombic form and 1.8 A for the monoclinic form. A platinum derivative was prepared for the orthorhombic and monoclinic crystals using K(2)PtCl(4) and data were collected at several wavelengths to obtain phases by the MAD technique. A partial model has been built from the electron-density maps of both forms and refinement is in progress.

Estudio parasitológico-entomológico de triatomíneos procedentes de la Provincia Zudañez, Departamento de Chuquisaca / Study of triatomineos at the Zudañes province Department of Chuquisaca

Durante el año de 1995, fue realizado un estudio entomológico-parasitológico en la Provincia Zudañez del Departamento de Chuquisaca, para identificar las especies de triatominos existentes, el grado de infestación en el domicilio y en las comunidades y el grado de infección de T. cruzi