The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress

Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ~35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48-61) large-scale proteolysis of the cytoskeleton during rupture ~48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release.     

The Trypanosoma cruzi protease cruzain mediates immune evasion

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ～P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.     

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.     

Mitochondria can recognize and assemble fragments of a beta-barrel structure

β-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial β-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full β-barrel structure is required, we expressed in yeast cells the β-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded β-barrel structure to which each monomer is contributing four β-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete β-barrel structure, four β-strands are sufficient for the mitochondria to recognize and assemble a β-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.     

Impact of Admission and Cache Replacement Policies on Response Times of Jobs on Data Grids

Caching techniques have been used widely to improve the performance gaps of storage hierarchies in computing systems. Little is known about the impact of policies on the response times of jobs that access and process very large files in data grids, particularly when data and computations on the data have to be co-located on the same host. In data intensive applications that access large data files over wide area network environment, such as data-grids, the combination of policies for job servicing (or scheduling), caching and cache replacement can significantly impact the performance of grid jobs. We present preliminary results of a simulation study that combines an admission policy with a cache replacement policy when servicing jobs submitted to a storage resource manager.The results show that, in comparison to a first come first serve policy, the response times of jobs are significantly improved, for practical limits of disk cache sizes, when the jobs that are back-logged to access the same files are taken into consideration in scheduling the next file to be retrieved into the disk cache. Not only are the response times of jobs improved, but also the metric measures for caching policies, such as the hit ratio and the average cost per retrieval, are improved irrespective of the cache replacement policy used. Ekow Otoo is research staff scientist with the scientific data management group at Lawrence Berkeley National Laboratory, University of California, Berkeley. He received his B.Sc. degree in Electrical Engineering from the University of Science and Technology, Kumasi, Ghana and a post graduate diploma in Computer Science from the University of Ghana, Legon. In 1977, he received his M.Sc. degree in Computer Science from the University of Newcastle Upon Tyne in Britain and his Ph.D. degree in Computer Science from McGill University, Montreal, Canada in 1983. He joined the faculty of the School of Computer Science, Carleton University, in 1983 and from 1987 to 1999, he was a tenured faculty member of the School of Computer Science, Carleton University, Ottawa, Canada. He has served as research consultant to Bell Northern Research, Ottawa, Canada, and as a research project consultant to the GIS Division, Geomatics Canada, Natural Resources Canada, from 1990 to 1998. Ekow Otoo is a member of the ACM and IEEE. His research interests include database management systems, data structures and algorithms, parallel I/O for high performance computing, parallel and distributed computing. Doron Rotem is currently a senior staff scientist and a member of the Data Management group at the Lawrence Berkeley National Lab. His research interests include Grid Computing, Workflow, Scientific Data Management and Paralled and Distributed Computing and Algorithms. He has published over 80 papers in international journals and conferences in these areas. Prior to that, Dr Rotem co-founded and served as a CTO of a startup company, called CommerceRoute, that made software products in the area of workflow and data integration and before that, he was an Associate Professor in the Department of Computer Science, University of Waterloo, Canada. Dr. Rotem holds a B.Sc degree in Mathematics and Statistics from the Hebrew University, Jerusalem, Israel and a Ph.D. in Computer Science from the University of the Witwatersrand, Johannesburg, South Africa. Arie Shoshani is a senior staff scientist at Lawrence Berkeley National Laboratory. He joined LBNL in 1976. He heads the Scientific Data Management Group. He received his Ph.D. from Princeton University in 1969. From 1969 to 1976, he was a researcher at System Development Corporation, where he worked on the Network Control Program for the ARPAnet, distributed databases, database conversion, and natural language interfaces to data management systems. His current areas of work include data models, query languages, temporal data, statistical and scientific database management, storage management on tertiary storage, and grid storage middleware. Arie is also the director of a Scientific Data Management (SDM) Integrated Software Infrastructure Center (ISIC), one of seven centers selected by the SciDAC program at DOE in 2001. In this capacity, he is coordinating the work of collaborators from 4 DOE laboratories and 4 universities (see: http://sdmcenter.lbl.gov). Dr. Shoshani has published over 65 technical papers in refereed journals and conferences, chaired several workshops, conferences, and panels in database management; and served on numerous program committees for various database conferences. He also served as an associate editor for the ACM Transactions on Database Systems. He was elected a member of the VLDB Endowment Board, served as the Publication Board Chairperson for the VLDB Journal, and as the Vice-President of the VLDB Endowment. His home page is http://www.lbl.gov/arie.     

Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement

Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neo-loxP cassette was placed under the control of the endogenous mouse beta-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) cells as directly in zygotes. Our results demonstrate ubiquitous expression of floxed transgenes in the endogenous beta-actin locus and they support the general use of the beta-actin locus for targeted transgenesis.     

Inhibition of HIV-1 envelope glycoprotein-mediated cell fusion by a DL-amino acid-containing fusion peptide: possible recognition of the fusion complex

The N-terminal fusion peptide (FP) of human immunodeficiency virus-1 (HIV-1) is a potent inhibitor of cell-cell fusion, possibly because of its ability to recognize the corresponding segments inside the fusion complex within the membrane. Here we show that a fusion peptide in which the highly conserved Ile(4), Phe(8), Phe(11), and Ala(14) were replaced by their d-enantiomers (IFFA) is a potent inhibitor of cell-cell fusion. Fourier transform infrared spectroscopy confirmed that despite these drastic modifications, the peptide preserved most of its structure within the membrane. Fluorescence energy transfer studies demonstrated that the diastereomeric peptide interacted with the wild type FP, suggesting this segment as the target site for inhibition of membrane fusion. This is further supported by the similar localization of the wild type and IFFA FPs to microdomains in T cells and the preferred partitioning into ordered regions within sphingomyelin/phosphatidyl-choline/cholesterol giant vesicles. These studies provide insight into the mechanism of molecular recognition within the membrane milieu and may serve in designing novel HIV entry inhibitors.