Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.     

Spectrophotometric assay for ornithine decarboxylase

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.     

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from ²H-hyperfine interaction with D₂O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D₂O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D₂O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D₂O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D₂O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.     

Analysis of the bilayer phase transition temperatures of phosphatidylcholines with mixed chains

The analysis of the chain-length dependence of the chain-melting transition temperatures of bilayers composed of lipids with identical chains (Marsh, D. 1991. Biochim. Biophys. Acta. 1062: 1-6) is extended to include lipids with chains of unequal length. The bilayer transition temperatures of saturated asymmetrical phosphatidylcholines are interpreted by assuming that the transition enthalpy and transition entropy are linearly related to the absolute value of the difference in chain length between the sn-1 and sn-2 chains, with constant end contributions. Such an assumption is supported by calorimetric data on phosphatidylcholines of constant mean chainlength and varying chain asymmetry. In particular, a symmetrical linear dependence is observed on the chain asymmetry, Δn, which is centered around a value Δn° that corresponds to the conformational inequivalence of the sn-1 and sn-2 chains. The transition temperature then takes the form: T_t = T_t^∞(n - n_H - h′ | Δn + Δn° |)/(n - n_s - s′ | Δn + Δn°) where n_H, n_s are the end contributions, and h′, s′ are fractional deficits in the incremental transition enthalpy and entropy, respectively, arising from the overlapping regions of the longer chains. Optimization on the transition temperature data for the dependence on chain asymmetry of three series of phosphatidylcholines with constant mean chainlength, n, yields parameters that are capable of predicting the dependence of the transition temperatures on chain asymmetry for other mean chainlengths. The dependence of the transition temperature on mean chainlength for phosphatidylcholines in which the chain asymmetry is maintained constant, as well as the dependence on both mean chain length and chain asymmetry for phosphatidylcholines in which one of the two chains is maintained of constant length, are also described with high accuracy by using the same parameters.     

Sequence of a novel HLA-DQB1 allele

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accesion number M74842. The name DQB1*0304 has been officially assigned by the WHO Nomenclature Committee in November 1991. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (WHO Nomenclature Committee for factors of the HLA system, 1991), names will be assigned to new sequences as they are identified. List of such new names will be published in the following WHO Nomenclature Report.     

Identification of a linear B-cell epitope on the Schistosoma japonicum saposin protein,SjSAP4: Potential as a component of a multi-epitope diagnostic assay

BackgroundSchistosoma japonicum is one of three major species of blood flukes causing schistosomiasis, a disease, which continues to be a major public health issue in the Philippines. SjSAP4, a member of a multigene family of saposin-like proteins, is a recognized immunodiagnostic biomarker for schistosomiasis japonica. This study aimed to identify linear B-cell epitopes on SjSAP4 and to validate their potential as components of a multi-epitope assay for the serological diagnosis of schistosomiasis japonica.MethodologySjSAP4-derived peptides were expressed as GST-peptide-fused proteins and these were Western blot probed with human serum samples from S. japonicum Kato-Katz (KK)-positive individuals and uninfected controls. A core epitope was further identified by Western blotting through probing a series of truncated peptides with the schistosomiasis patient sera. The diagnostic performance of the core epitope-containing peptides and the full-length SjSAP4 was evaluated by enzyme-linked immunosorbent assay (ELISA) using a panel of sera collected from subjects resident in a schistosomiasis-endemic area of the Philippines.Main findingsAs a result of the peptide mapping, one peptide (P15) was found to be highly immunogenic in the KK-positive individuals. We subsequently showed that -S¹⁶³QCSLVGDIFVDKYLD¹⁷⁸- is a core B-cell epitope of P15. Subsequent ELISAs incorporating SjSAP4, SjSAP4-Peptide and SjSP-13V2-Peptide showed a sensitivity of 94.0%, 46.0% and 74.0%, respectively, and a specificity of 97.1%, 100% and 100%, respectively. Notably, complementary recognition of the B-cell epitopes (SjSAP4-Peptide and SjSP-13V2-Peptide) was observed in a subset of the KK-positive individuals. A dual epitope-ELISA (SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA) showed a diagnostic sensitivity of 84.0% and a specificity of 100%.Conclusions/SignificanceIn this study, -S¹⁶³QCSLVGDIFVDKYLD¹⁷⁸- was identified as a dominant linear B-cell epitope on SjSAP4. This peptide and the complementary recognition of other B-cell epitopes using sera from different KK-positive individuals can provide the basis of developing a multi-epitope assay for the serological diagnosis of schistosomiasis.     

Ecological dynamics of emerging bat virus spillover

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.     

Benefits in Cash or in Kind? A Community Consultation on Types of Benefits in Health Research on the Kenyan Coast

BackgroundProviding benefits and payments to participants in health research, either in cash or in kind, is a common but ethically controversial practice. While much literature has concentrated on appropriate levels of benefits or payments, this paper focuses on less well explored ethical issues around the nature of study benefits, drawing on views of community members living close to an international health research centre in Kenya.MethodsThe consultation, including 90 residents purposively chosen to reflect diversity, used a two-stage deliberative process. Five half-day workshops were each followed by between two and four small group discussions, within a two week period (total 16 groups). During workshops and small groups, facilitators used participatory methods to share information, and promote reflection and debate on ethical issues around types of benefits, including cash, goods, medical and community benefits. Data from workshop and field notes, and voice recordings of small group discussions, were managed using Nvivo 10 and analysed using a Framework Analysis approach.

Findings and Conclusions

The methods generated in-depth discussion with high levels of engagement. Particularly for the most-poor, under-compensation of time in research carries risks of serious harm. Cash payments may best support compensation of costs experienced; while highly valued, goods and medical benefits may be more appropriate as an ‘appreciation’ or incentive for participation. Community benefits were seen as important in supporting but not replacing individual-level benefits, and in building trust in researcher-community relations. Cash payments were seen to have higher risks of undue inducement, commercialising relationships and generating family conflicts than other benefits, particularly where payments are high. Researchers should consider and account for burdens families may experience when children are involved in research. Careful context-specific research planning and skilled and consistent communication about study benefits and payments are important, including in mitigating potential negative effects.