Organization of ETRAMPs and EXP-1 at the parasite-host cell interface of malaria parasites

The parasite-host cell interface is a key compartment of vacuolated intracellular pathogens but little is known about its molecular composition and architecture. We used in vivo cross-linking to analyse the parasite-host cell interface of asexual stages of the most virulent human malaria parasite Plasmodium falciparum. We show that the integral membrane protein members of the early transcribed membrane protein (ETRAMP) family and exported protein 1 (EXP-1), which are components of the parasite-host cell interface, form complexes of oligomeric arrays in this compartment. The most notable feature is that each ETRAMP member and EXP-1 define separate arrays, demonstrating that the protein distribution in this membrane is non-random. Each of three recombinant ETRAMPs readily oligomerized in bacterial membranes, confirming that these arrays can form independently of other Plasmodium proteins. We propose that the malaria parasite-host cell interface contains patches of integral membrane proteins forming a mosaic of different microdomains in this membrane.     

Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.     

Standardising bull breeding soundness evaluations and reporting in Australia

There is substantial variation in bull breeding soundness evaluation procedures and reports in Australia; the situation is compounded by difficulties in interpretation and the validity of many reports. In an effort to overcome this, the scientific literature was reviewed [Fordyce G. In: Fordyce G, editor. Bull fertility: selection and management in Australia. Eight Mile Plains, Australia: Australian Cattle Vets; 2002] and the needs of stakeholders were considered in preparing a manual, Evaluating and Reporting Bull Fertility [Entwistle KW, Fordyce G. Evaluating and reporting bull fertility. Eight Mile Plains, Australia: Australian Cattle Vets; 2003.] that outlined standards for assessing and reporting bull breeding soundness. A new recording and reporting system, called Bull Reporter, is based on standards from this manual and groups bull fertility traits into five summary categories: Scrotum, Physical, Crush-side Semen, Sperm Morphology, and Serving. The client will generally select which categories they wish to have included in the evaluation to suit their specific purposes. While there is adequate room for comments, the veterinarian is not required to make an overall judgment of whether the bull has normal capacity to sire calves under natural mating management, but ensures the standards for each selected category are met. Professional, standardised, easy-to-read reports are produced either electronically [Entwistle KW, Fordyce G. Evaluating and reporting bull fertility. Eight Mile Plains, Australia: Australian Cattle Vets; 2003.] or manually. A bull owner or their agent signs the certificate to affirm that bulls have not undergone procedures to rectify faults which may have otherwise caused them to fail the standards. An accreditation system for assessing sperm morphology was established because of its demonstrated relationship with pregnancy rates and because of the difficulties in achieving consistent and accurate assessments among laboratories. It is considered that Bull Reporter is applicable to beef and dairy bulls across all levels of management, genotypes and environments throughout Australia, with substantial potential for application elsewhere in the world.     

Interaction of alpha-and beta-oligoarginine-acids and amides with anionic lipid vesicles: a mechanistic and thermodynamic study

The interaction of alpha- and beta-oligoarginine amides and acids and of alpha-polyarginine with anionic lipid vesicles was studied. The beta-oligoarginines used were beta3-homologues of the alpha-oligoarginines. Lipid bilayers were composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) containing 5 mol % pyrene-PG (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-[phospho-rac-1-glycerol]). Kinetic analysis of the binding process onto large unilamellar POPC/POPG (3:7, molar ratio) vesicles (100 nm diameter) shows biphasic time courses for all tested peptides. The first binding step is fast and takes place within approximately 10 s with no disruption of the membrane as indicated by corresponding calcein release measurements. The second binding phase is slow and occurs within the next 30-300 s with substantial membrane disruption. In this context, beta-hexa- and octaarginine amides possess higher second half-times than the beta-hexa- and octaarginine acids of the same chain length. Furthermore beta-octaarginine amide induces a calcein release approximately twice as large as that of the beta-octaarginine acid. Thermodynamic analysis of the binding process, using the complex formation model that assumes that each peptide binds independently to n POPG lipids, reveals apparent binding constants (K(app1)) of approximately 5 x 10(6)-10(8) M(-1) and n-values from 3.7 for beta-hexaarginine acid up to 24.8 for alpha-polyarginine. Although the K(app1)-values are similar, the number of binding sites clearly depends on the chemical nature of the oligoarginine: beta-oligoarginine amides and alpha-oligoarginine acids interact with more lipids than beta-oligoarginine acids of the same length. Calculation of the electrostatic contribution to the total free energy of binding reveals that for all oligoarginines only 25-30% has electrostatic origin. The remaining approximately 70-75% is nonelectrostatic, corresponding to hydrogen bonding and/or hydrophobic interactions. From the obtained data, a mechanism is suggested by which oligoarginines interact with anionic vesicles: (1) initial electrostatic interaction that is fast, nonspecific, and relatively weak; (2) nonelectrostatic interaction that is rate-limiting, stronger, and induces bilayer rigidification as well as release of aqueous contents from the vesicles.     

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.     

A case of 'hit-and-run' in Plasmodium genetics

The ability to genetically manipulate species of the genus Plasmodium, some of which are causative organisms of malaria, has seen significant advances in the past 13 years. However, one major tool that has been lacking is the ability to undertake reverse genetics and 'hit-and-run' mutagenesis. This deficiency has been addressed in the Plasmodium berghei model.     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.     

A highly conserved effector in Fusarium oxysporum is required for full virulence on Arabidopsis

Secreted-in-xylem (SIX) proteins of the vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici are secreted during infection of tomato and function in virulence or avirulence. F. oxysporum formae speciales have specific host ranges but the roles of SIX proteins in diverse hosts are unknown. We identified homologs of F. oxysporum f. sp. lycopersici SIX1, SIX4, SIX8, and SIX9 in the genome of Arabidopsis infecting isolate Fo5176. A SIX4 homolog (termed Fo5176-SIX4) differed from that of F. oxysporum f. sp. lycopersici (Fol-SIX4) by only two amino acids, and its expression was induced during infection of Arabidopsis. Transgenic Arabidopsis plants constitutively expressing Fo5176-SIX4 had increased disease symptoms with Fo5176. Conversely, Fo5176-SIX4 gene knock-out mutants (Δsix4) had significantly reduced virulence on Arabidopsis, and this was associated with reduced fungal biomass and host jasmonate-mediated gene expression, the latter known to be essential for host symptom development. Full virulence was restored by complementation of Δsix4 mutants with either Fo5176-SIX4 or Fol-SIX4. Thus, Fo5176-SIX4 contributes quantitatively to virulence on Arabidopsis whereas, in tomato, Fol-SIX4 acts in host specificity as both an avirulence protein and a suppressor of other race-specific resistances. The strong sequence conservation for SIX4 in F. oxysporum f. sp. lycopersici and Fo5176 suggests a recent common origin.     

X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.