Identification of Plasmodium Falciparum proteins interacting with the erythrocyte membrane skeleton protein spectrin

Malaria, which is caused by Plasmodium parasites, is responsible for the death of millions of humans every year in the tropical and subtropical regions of the world. Specifically P. falciparum, one of four malaria species infecting humans, is responsible for the greatest morbidity and mortality burden in African populations. The Anopheles mosquito transmits the parasite to the human host, where it infects and destroys human erythrocytes. The erythrocyte membrane therefore plays a vital role in all aspects of the pathogenic phase of the P. falciparum life cycle and protein-protein interactions between host and parasite are thus a key focus of research. The human erythrocyte maintains its shape with a structural network of proteins underneath the plasma membrane and the main protein component of this erythrocyte membrane skeleton is spectrin. To investigate host-parasite protein interactions, a novel application of phage display technology was developed, whereby purified human erythrocyte spectrin was biopanned against a P. falciparum phage-display library. The P. falciparum DNA inserts of interacting phage were compared to the PlasmoDB database and five interacting proteins were identified: a putative aminopeptidase (PfM18AAP); a putative Ebl-1 like protein, which is proposed to participate in erythrocyte invasion; and three hypothetical proteins. The interaction of the hypothetical proteins with spectrin is the first information available on the function of these proteins. The five gene sequences were cloned into the pET-15b or pGEX-4T-2 expression vectors for purification of the recombinant proteins from Escherichia coli. Only the 6His-PfM18AAP fusion protein was expressed in soluble form and purified by affinity selection. PfM18AAP migrated as a 67 kDa peptide on SDS-PAGE and native gel analysis revealed multiple subunits of the enzyme, predominantly a tetramer and higher oligomers. Cleavage of the 6His-tag and subsequent IEF SDS-PAGE revealed three 65 kDa entities with pI ~6.6, ~6.7 and ~6.9. An in vitro coupled enzyme assay showed that PfM18AAP cleaved an N-terminal aspartate from a peptide substrate with a maximum activity at pH 7.5 and 37 ºC. Inhibitor studies confirmed that the enzyme is a metalloprotease. Blot overlay assays with PfM18AAP against spectrin and erythrocyte membrane proteins verified that PfM18AAP binds strongly to β-spectrin, as well as protein 4.1, protein 4.2, actin and glyceraldehyde-3-phosphate dehydrogenase. Comparison of the PfM18AAP protein sequence to ten other M18 aminopeptidase sequences, including human and three other Plasmodium species, revealed that all the critical amino acids responsible for the binding of two catalytic metal ions, enzymatic catalysis and quaternary structure stabilisation are conserved. The peptide fragment, which initially bound to spectrin during phage display, is not found in other M18 aminopeptidases, suggesting that the presence of this fragment is an evolutionary development of P. falciparum that allows the protease to bind to human spectrin. Analysis of four M18 aminopeptidase crystal structures revealed that the spectrin-binding region forms an external loop on the protein and would thus be accessible to spectrin. Results from this study suggest that, apart from haemoglobin digestion, PfM18AAP performs additional functions in the parasite and infected erythrocyte by cleaving spectrin and other erythrocyte membrane proteins. This would destabilise and disrupt the erythrocyte membrane skeleton to facilitate entry or exit from the host cell, or the insertion of parasite proteins into the host cell membrane. Further analysis and characterisation of PfM18AAP and its interactions with the erythrocyte membrane proteins will shed more light on the multifunctional role of this parasite enzyme. Studies of this enzyme and the hypothetical proteins may also aid in the quest to discover new therapeutics to combat this killer disease

Whole-body MRI of multiple myeloma: Comparison of different MRI sequences in assessment of different growth patterns

Die MRT hat die höchste Detektionsrate der radiologischen Verfahren zur Erkennung einer Knochenmarksinfiltration durch das multiple Myelom. In der vorliegenden Studie hatte die T2-w TIRM Sequenz die höchste Sensitivität. Es konnte jedoch kein signifikanter Unterschied zwischen der T2w-TIRM Sequenz und der T1-w TSE Sequenz festgestellt werden. Die Interobservervarianz war in Sitzung 2 (T2-w TIRM Sequenz) und Sitzung 3 (T2-w TIRM/T1-w TSE/KM +T1-w TSE Sequenz) gleichwertig. Durch Kontrastmittelgabe konnte die Sensitivität nicht signifikant verbessert werden. Zusammenfassend bietet die T2-w TIRM Sequenz die Möglichkeit einer ersten schnellen orientierenden Untersuchung. Für ein detailliertes radiologisches Staging, Grading und zum Therapiemonitoring sollte jedoch weiterhin eine MRT-Untersuchung mit T2-w TIRM/T1-w TSE Sequenz plus zusätzlicher Kontrastmittelzugabe erfolgen.For detecting bone marrow plasma cell infilration by multiple myeloma MR imaging ist superior to other imaging techniques. The following study shows that T2w-TIRM sequences achieved the highest level of sensitivity and the best reliability depending on infiltration grade and clinical stage. Differences between T2-w TIRM and T1w-TSE sequence are not significant. The interobserver variability was same in reading 2 (T2w-TIRM sequence) and reading 3 (T2w-TIRM/T1-w TSE/KM+ T1-w TSE sequence). Contrast-enhanced sequences could not significantly improve sensitivity. Conclusionally T2w-TIRM sequences might be valuable for initial assessment of MM. For an exact staging, grading and therapy monitoring the examination protocol should encompass unenhanced and enhanced T1w- MRI sequences in addition to T2w-TIRM

Whole-body MRI of multiple myeloma: comparison of different MRI sequences in assessment of different growth patterns

PURPOSE: To determine sensitivity, specificity and inter-observer variability of different whole-body MRI (WB-MRI) sequences in patients with multiple myeloma (MM). METHODS AND MATERIALS: WB-MRI using a 1.5T MRI scanner was performed in 23 consecutive patients (13 males, 10 females; mean age 63+/-12 years) with histologically proven MM. All patients were clinically classified according to infiltration (low-grade, n=7; intermediate-grade, n=7; high-grade, n=9) and to the staging system of Durie and Salmon PLUS (stage I, n=12; stage II, n=4; stage III, n=7). The control group consisted of 36 individuals without malignancy (25 males, 11 females; mean age 57+/-13 years). Two observers independently evaluated the following WB-MRI sequences: T1w-TSE (T1), T2w-TIRM (T2), and the combination of both sequences, including a contrast-enhanced T1w-TSE with fat-saturation (T1+/-CE/T2). They had to determine growth patterns (focal and/or diffuse) and the MRI sequence that provided the highest confidence level in depicting the MM lesions. Results were calculated on a per-patient basis. RESULTS: Visual detection of MM was as follows: T1, 65% (sensitivity)/85% (specificity); T2, 76%/81%; T1+/-CE/T2, 67%/88%. Inter-observer variability was as follows: T1, 0.3; T2, 0.55; T1+/-CE/T2, 0.55. Sensitivity improved depending on infiltration grade (T1: 1=60%; 2=36%; 3=83%; T2: 1=70%; 2=71%; 3=89%; T1+/-CE/T2: 1=50%; 2=50%; 3=89%) and clinical stage (T1: 1=58%; 2=63%; 3=79%; T2: 1=58%; 2=88%; 3=100%; T1+/-CE/T2: 1=50%; 2=63%; 3=100%). T2w-TIRM sequences achieved the best reliability in depicting the MM lesions (65% in the mean of both readers). CONCLUSIONS: T2w-TIRM sequences achieved the highest level of sensitivity and best reliability, and thus might be valuable for initial assessment of MM. For an exact staging and grading the examination protocol should encompass unenhanced and enhanced T1w-MRI sequences, in addition to T2w-TIRM