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

Comparison of immune response generated against Japanese encephalitis virus envelope protein expressed by DNA vaccines under macrophage associated versus ubiquitous expression promoters

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis, with ~50,000 cases reported annually worldwide. Vaccination is the only measure for prevention. Recombinant vaccines are an efficient and safe alternative for formalin inactivated or live attenuated vaccines. Nowadays, incorporation of molecular adjuvants has been the main strategy for melioration of vaccines. Our attempt of immunomodulation is based on targeting antigen presenting cells (APC) "majorly macrophages" by using macrosialin promoter. We have compared the immune response of the constructed plasmids expressing JEV envelope (E) protein under the control of aforesaid promoter and cytomegalovirus (CMV) immediate early promoter in mouse model. Protection of immunized mice from lethal challenge with JEV was also studied.</p> <p>Results</p> <p>The E protein was successfully expressed in the macrophage cell line and was detected using immunofluorescence assay (IFA) and Western blotting. APC expressing promoter showed comparable expression to CMV promoter. Immunization of mice with either of the plasmids exhibited induction of variable JEV neutralizing antibody titres and provided protection from challenge with a lethal dose of JEV. Immune splenocytes showed proliferative response after stimulation with the JEV antigen (Ag), however, it was higher for CMV promoter. The magnitude of immunity provided by APC dominant promoter was non-significantly lower in comparison to CMV promoter. More importantly, immune response directed by APC promoter was skewed towards Th1 type in comparison to CMV promoter, this was evaluated by cytokine secretion profile of immune splenocytes stimulated with JEV Ag.</p> <p>Conclusions</p> <p>Thus, our APC-expressing DNA vaccination approach induces comparable immunity in comparison to ubiquitous promoter construct. The predominant Th1 type immune responses provide opportunities to further test its potency suitable for response in antiviral or anticancer vaccines.</p

Does type of hospital ownership influence physicians' daily work schedules? An observational real-time study in German hospital departments

Background: During the last two decades the German hospital sector has been engaged in a constant process of transformation. One obvious sign of this is the growing amount of hospital privatization. To date, most research studies have focused on the effects of privatization regarding financial outcomes and quality of care, leaving important organizational issues unexplored. Yet little attention has been devoted to the effects of privatization on physicians' working routines. The aim of this observational real-time study is to deliver exact data about physicians' work at hospitals of different ownership. By analysing working hours, further impacts of hospital privatization can be assessed and areas of improvement identified. Methods: Observations were made by shadowing 100 physicians working in private, for-profit or non-profit as well as public hospital departments individually during whole weekday shifts in urban German settings. A total of 300 days of observations were conducted. All working activities were recorded, accurate to the second, by using a mobile personal computer. Results: Results have shown significant differences in physicians' working activities, depending on hospital ownership, concerning working hours and time spent on direct and indirect patient care. Conclusion: This is the first real-time analysis on differences in work activities depending on hospital ownership. The study provides an objective insight into physicians' daily work routines at hospitals of different ownership, with additional information on effects of hospital privatization