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

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses

The human capital transition and the role of policy

Along with information and communication technology, infrastructure, and the innovation system, human capital is a key pillar of the knowledge economy with its scope for increasing returns. With this in mind, the purpose of this chapter is to investigate how industrialized economies managed to achieve the transition from low to high levels of human capital. The first phase of the human capital transition was the result of the interaction of supply and demand, triggered by technological change and boosted by the demands for (immaterial) services. The second phase of the human capital transition (i.e., mass education) resulted from enforced legislation and major public investment. The state’s aim to influence children’s beliefs appears to have been a key driver in public investment. Nevertheless, the roles governments played differed according to the developmental status and inherent socioeconomic and political characteristics of their countries. These features of the human capital transition highlight the importance of understanding governments’ incentives and roles in transitions

Human malarial disease: a consequence of inflammatory cytokine release

Malaria causes an acute systemic human disease that bears many similarities, both clinically and mechanistically, to those caused by bacteria, rickettsia, and viruses. Over the past few decades, a literature has emerged that argues for most of the pathology seen in all of these infectious diseases being explained by activation of the inflammatory system, with the balance between the pro and anti-inflammatory cytokines being tipped towards the onset of systemic inflammation. Although not often expressed in energy terms, there is, when reduced to biochemical essentials, wide agreement that infection with falciparum malaria is often fatal because mitochondria are unable to generate enough ATP to maintain normal cellular function. Most, however, would contend that this largely occurs because sequestered parasitized red cells prevent sufficient oxygen getting to where it is needed. This review considers the evidence that an equally or more important way ATP deficency arises in malaria, as well as these other infectious diseases, is an inability of mitochondria, through the effects of inflammatory cytokines on their function, to utilise available oxygen. This activity of these cytokines, plus their capacity to control the pathways through which oxygen supply to mitochondria are restricted (particularly through directing sequestration and driving anaemia), combine to make falciparum malaria primarily an inflammatory cytokine-driven disease