The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro

<p>Abstract</p> <p>Background</p> <p>During erythrocytic schizogony, <it>Plasmodium falciparum </it>interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the <it>P. falciparum </it>M18 aspartyl aminopeptidase (<it>Pf</it>M18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) <it>Pf</it>M18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.</p> <p>Methods</p> <p>r<it>Pf</it>M18AAP was produced as a hexahistidine-fusion protein in <it>Escherichia coli </it>and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-<it>Pf</it>M18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.</p> <p>Results</p> <p>r<it>Pf</it>M18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that r<it>Pf</it>M18AAP aggregated into oligomers. An <it>in vitro </it>coupled enzyme assay showed that r<it>Pf</it>M18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37°C. The spectrin-binding region of <it>Pf</it>M18AAP is not found in <it>Homo sapiens, Saccharomyces cerevisiae </it>and other<it>Plasmodium </it>species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with r<it>Pf</it>M18AAP against spectrin and erythrocyte membrane proteins indicated that r<it>Pf</it>M18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.</p> <p>Conclusion</p> <p>Studies characterizing r<it>Pf</it>M18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, <it>Pf</it>M18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.</p

UV-induced effects

Regulatory requirements: According to the current Notes for Guidance of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, for example, to ensure the light stability of cosmetics or used in sun protection products) should be tested for acute phototoxic and photogenotoxic potential. Testing for photosensitisation (immunological photoallergy) potential is not specifically required, but it is nevertheless often performed. Acute phototoxicity: Due to a thorough multi-stage and multi-centre validation trial (1992-1998) the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) had already gained acceptance by the SCCNFP in 1998, and it is recommended by the EMEA/CPMP as a basic preclinical test for acute phototoxicity. It was accepted as Method No. 41 in Annex V to Directive 67/548/EEC in the year 2000, and was accepted as the new Test Guideline 432 by the OECD in 2002. The 3T3-NRU-PT is regarded as a basic screen for identifying acute phototoxic potential. Two additional in vitro tests, formally evaluated in controlled blind trials, the RBC Phototoxicity Test (RBC-PT) and the Human 3-D Skin Model Phototoxicity Test (H3D-PT), are regarded as useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the RBC-PT permits an evaluation of the phototoxic mechanisms involved. In conclusion, the identification of acute phototoxic hazards is now regarded as being sufficiently covered by in vitro tests, so that animal testing for that endpoint can now be 100% replaced. Photogenotoxicity: In the area of photogenotoxicity, almost the whole battery of in vitro genetic toxicity tests have been (or are currently being) converted into test protocols of photogenotoxicity tests. Tests exclusively predictive for gene mutation, for example, the Photo-Ames (P-Ames) Test and the Photo-Thymidine Kinase Test (P-TKT), have become less important than tests for clastogenic effects (for example, the Photo-Chromosome Aberration Test [P-CAT] and the Photo-Micronucleus Test [P-MNT]). In addition, a number of promising indicator tests, such as the Photo-Comet Assay (P-Comet) have been developed. Although routinely used, to date none of the new photogenotoxicity tests have been formally validated. Therefore, the P-MNT and the P-Comet are currently being evaluated in a formal interlaboratory validation study. It is expected that these in vitro photogenotoxicity test methods may become available as validated and accepted methods within the next five years. Photoallergy (Photosensitisation): In the area of photoallergy (photosensitisation), as development of predictive in vitro tests for delayed contact sensitisation (allergenicity) potential without the involvement of light, due to a lack of ability to model the complex mechanisms underlying allergy, no promising in vitro methods to predict photo-sensitisation potential are currently in sight (see the section on skin sensitisation). One in vitro screening method, which models the covalent binding of a light activated chemical to human serum albumin, may become relevant. However, while the binding of an excited chemical to proteins is a prerequisite for photoallergy, this is not a sufficient predictor on its own. The only promising alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (PLLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of "dark" sensitisation potential have been developed and accepted, their adaptation into similar photosensitisation testing will become possible

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

Summary: Bartonella spp. are facultative intracellular pathogens that employ a unique stealth infection strategy comprising immune evasion and modulation, intimate interaction with nucleated cells, and intraerythrocytic persistence. Infections with Bartonella are ubiquitous among mammals, and many species can infect humans either as their natural host or incidentally as zoonotic pathogens. Upon inoculation into a naive host, the bartonellae first colonize a primary niche that is widely accepted to involve the manipulation of nucleated host cells, e.g., in the microvasculature. Consistently, in vitro research showed that Bartonella harbors an ample arsenal of virulence factors to modulate the response of such cells, gain entrance, and establish an intracellular niche. Subsequently, the bacteria are seeded into the bloodstream where they invade erythrocytes and give rise to a typically asymptomatic intraerythrocytic bacteremia. While this course of infection is characteristic for natural hosts, zoonotic infections or the infection of immunocompromised patients may alter the path of Bartonella and result in considerable morbidity. In this review we compile current knowledge on the molecular processes underlying both the infection strategy and pathogenesis of Bartonella and discuss their connection to the clinical presentation of human patients, which ranges from minor complaints to life-threatening disease