Juxtamembrane shedding of Plasmodium falciparum AMA1 is sequence independent and essential, and helps evade invasion-inhibitory antibodies.

The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However - in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.

Ultrasound assessment of bilateral symmetry in dorsal Lisfranc ligament.

Bilateral symmetry of the ligaments is a common assumption used as an intrasubject control for clinical diagnosis. The present study investigated the bilateral symmetry of the dorsal Lisfranc ligament (dLL) using ultrasound. Data were acquired from 50 asymptomatic subjects in a seated position at a loaded calf raise machine equipped with a force plate. The testing conditions included low, medium, and high stress at 0° and 15° abducted foot positions. Images of the dLL were captured and measured using a 10.0-MHz ultrasound transducer and custom written MATLAB software, respectively. The data were analyzed using paired t tests to compare the bilateral measurements of the dLL length under all test conditions. The bilateral pooled dLL length was 7.01 ± 1.38 mm and showed a moderate correlation with the foot length and width. No bilateral differences were found in the dLL length under any of the stress loads in the abducted position or under the medium and high stress load in the rectus position. However, the low stress load rectus position demonstrated a significant bilateral difference in the dLL length (p = .005). The smallest bilateral difference was observed at the 15° abducted position under medium stress (measurement error mean -0.062 mm). Our data suggest that the contralateral dLL length can be used as an intrasubject control for clinical purposes. However, we recommend that the dLL length measurements should be taken in weightbearing position with the foot in the abducted position under medium stress (bilateral stance), reducing potential strain-induced asymmetry.

Structural basis for alpha-conotoxin potency and selectivity.

Parkinson's disease is a debilitating movement disorder characterized by altered levels of alpha(6)beta(2) * ( * indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. alpha-Conotoxin MII (alpha-CTx MII) is a highly useful ligand to probe alpha(6)beta(2) nAChRs structure and function, but it does not discriminate among closely related alpha(6) * nAChR subtypes. Modification of the alpha-CTx MII primary sequence led to the identification of alpha-CTx MII[E11A], an analog with 500-5300-fold discrimination between alpha(6) * subtypes found in both human and non-human primates. alpha-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity alpha(6) nAChR, a subtype that is selectively lost in Parkinson's disease. Here, we present the three-dimensional solution structure for alpha-CTx MII[E11A] as determined by two-dimensional (1)H NMR spectroscopy to 0.13+/-0.09A backbone and 0.45+/-0.08A heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of alpha-CTx MII[E11A] relative to other members of the alpha-CTx family may be responsible for its exceptionally high affinity for alpha6alpha4beta2 * nAChR as well as discrimination between alpha(6)beta(2) and alpha(3)beta(2) containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for alpha(6) * nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson's disease.

Ultrasound assessment of dorsal lisfranc ligament strain under clinically relevant loads.

BACKGROUND: Pure Lisfranc ligament injuries have a varied clinical presentation, making them difficult to diagnose. This study seeks to understand in vivo strain characteristics of the dorsal Lisfranc ligament under clinically relevant stress loads and foot orientations measured by ultrasound. METHODS: Randomized ultrasound imaging trials were performed on 50 asymptomatic feet of 20-to-32-year-old individuals who were free of lower-extremity abnormalities. The dorsal Lisfranc ligament was ultrasound imaged under low, medium, and high stress while at 0° and 15° abducted foot orientations. Load was applied using a seated calf-raise apparatus, and a single examiner performed all of the tests. Two-way repeated-measures analysis of variance was used to determine any significant load or position main effects or load × position interaction. RESULTS: Position main effect for dorsal Lisfranc ligament length demonstrated a significant overall increase in ligament length of 0.21 mm (P < .001), which reflects a 4.03% change in ligament length between the rectus and 15° abducted orientations. Furthermore, low and medium loads demonstrated significant length increase with position effect (P = .03 and P < .001, respectively). No significant load main effect or interaction was determined. CONCLUSIONS: Dorsal Lisfranc ligament length undergoes more strain in an abducted foot position at the same load compared with in a rectus foot. We advocate measuring under a medium load if possible and comparing foot positions for the maximum length changes. The participant stress loads and foot positions used are clinically feasible, which makes it possible to perform this ultrasound procedure in the clinical setting.

Reliability of ultrasound imaging in the assessment of the dorsal Lisfranc ligament.

BACKGROUND: The Lisfranc ligament plays an integral role in providing stability to the midfoot. Variable clinical presentations and radiographic findings make injuries to the Lisfranc ligament notoriously difficult to diagnose. Currently, radiographic evaluation is the mainstay in imaging such injuries; however, ultrasound has been suggested as a viable alternative. The objective of this study was to evaluate the intra-rater and inter-rater reliability in the measurement of the length of the dorsal Lisfranc ligament using ultrasound imaging in healthy, asymptomatic subjects. METHODS: The dorsal Lisfranc ligaments of fifty asymptomatic subjects (n = 100 feet) were imaged using a Siemens SONOLINE Antares Ultrasound Imaging System© under low, medium, and high stress loads at 0° and 15° abducted foot positions. The lengths of the ligaments were measured, and Interclass correlation coefficients were used to calculate within-session intra-rater reliability (n = 100 feet) as well as between-session intra-rater reliability (n = 40 feet) and between-session inter-rater reliability (n = 40 feet). RESULTS: The within-session intra-rater reliability results for dorsal Lisfranc ligament length had an average ICC of 0.889 (min 0.873 max 0.913). The average ICC for between-session intra-rater reliability was 0.747 (min 0.607 max 0.811). The average ICC for between-session inter-rater reliability was 0.685 (min 0.638 max 0.776). CONCLUSIONS: The measurement of the dorsal Lisfranc ligament length using ultrasound imaging shows substantial to almost perfect reliability when evaluating asymptomatic subjects. This imaging modality methodology shows promise and lays the foundation for further work in technique development towards the diagnostic identification of pathology within the Lisfranc ligament complex.

Phosphoserines on maize CENTROMERIC HISTONE H3 and histone H3 demarcate the centromere and pericentromere during chromosome segregation.

We have identified and characterized a 17- to 18-kD Ser50-phosphorylated form of maize (Zea mays) CENTROMERIC HISTONE H3 (phCENH3-Ser50). Immunostaining in both mitosis and meiosis indicates that CENH3-Ser50 phosphorylation begins in prophase/diplotene, increases to a maximum at prometaphase-metaphase, and drops during anaphase. Dephosphorylation is precipitous (approximately sixfold) at the metaphase-anaphase transition, suggesting a role in the spindle checkpoint. Although phCENH3-Ser50 lies within a region that lacks homology to any other known histone, its closest counterpart is the phospho-Ser28 residue of histone H3 (phH3-Ser28). CENH3-Ser50 and H3-Ser28 are phosphorylated with nearly identical kinetics, but the former is restricted to centromeres and the latter to pericentromeres. Opposing centromeres separate in prometaphase, whereas the phH3-Ser28-marked pericentromeres remain attached and coalesce into a well-defined tether that binds the centromeres together. We propose that a centromere-initiated wave of histone phosphorylation is an early step in defining the two major structural domains required for chromosome segregation: centromere (alignment, motility) and pericentromere (cohesion).

Centromeric retroelements and satellites interact with maize kinetochore protein CENH3.

Maize centromeres are composed of CentC tandem repeat arrays, centromeric retrotransposons (CRs), and a variety of other repeats. One particularly well-conserved CR element, CRM, occurs primarily as complete and uninterrupted elements and is interspersed thoroughly with CentC at the light microscopic level. To determine if these major centromeric DNAs are part of the functional centromere/kinetochore complex, we generated antiserum to maize centromeric histone H3 (CENH3). CENH3, a highly conserved protein that replaces histone H3 in centromeres, is thought to recruit many of the proteins required for chromosome movement. CENH3 is present throughout the cell cycle and colocalizes with the kinetochore protein CENPC in meiotic cells. Chromatin immunoprecipitation demonstrates that CentC and CRM interact specifically with CENH3, whereas knob repeats and Tekay retroelements do not. Approximately 38 and 33% of CentC and CRM are precipitated in the chromatin immunoprecipitation assay, consistent with data showing that much, but not all, of CENH3 colocalizes with CentC.