Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.     

The evolution of the Cercopithecini: a (post)modern synthesis

The Cercopithecini, or African guenon monkeys, are one of the most diverse clades of living primates and comprise the most species‐rich clade of Catarrhini. Species identity is announced by flamboyant coloration of the facial and genital regions and, more cryptically, by vigorous chromosomal rearrangements among taxa. Beneath the skin, however, these animals are skeletally conservative and show low levels of genetic sequence divergence consonant with recent divergence between congeneric species. The guenons clearly demonstrate that morphological, cytogenetic, and reproductive differentiation proceed at different rates during speciation. We review diverse kinds of data in an effort to understand this conundrum.     

Monitoring changing toxigenicity of a cyanobacterial bloom by molecular methods

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the beta and alpha subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.     

Replication blocking lesions present a unique substrate for homologous recombination

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.     

Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World

A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.     

GLD-3 and control of the mitosis/meiosis decision in the germline of Caenorhabditis elegans

Germ cells can divide mitotically to replenish germline tissue or meiotically to produce gametes. In this article, we report that GLD-3, a Caenorhabditis elegans Bicaudal-C homolog, promotes the transition from mitosis to meiosis together with the GLD-2 poly(A) polymerase. GLD-3 binds GLD-2 via a small N-terminal region present in both GLD-3S and GLD-3L isoforms, and GLD-2 and GLD-3 can be co-immunoprecipitated from worm extracts. The FBF repressor binds specifically to elements in the gld-3S 3′-UTR, and FBF regulates gld-3 expression. Furthermore, FBF acts largely upstream of gld-3 in the mitosis/meiosis decision. By contrast, GLD-3 acts upstream of FBF in the sperm/oocyte decision, and GLD-3 protein can antagonize FBF binding to RNA regulatory elements. To address the relative importance of these two regulatory mechanisms in the mitosis/meiosis and sperm/oocyte decisions, we isolated a deletion mutant, gld-3(q741), that removes the FBF-binding site from GLD-3L, but leaves the GLD-2-binding site intact. Animals homozygous for gld-3(q741) enter meiosis, but are feminized. Therefore, GLD-3 promotes meiosis primarily via its interaction with GLD-2, and it promotes spermatogenesis primarily via its interaction with FBF.     

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.     

Growth of choroid plexus epithelium vesicles in vitro depends on secretory activity

Although a number of models have been used to study choroid plexus epithelium (CPe) function, analysis in physiological conditions of this polarised epithelium which produces the majority of the cerebrospinal fluid (CSF) and is one of the key barriers between blood and CSF in the brain remains challenging. As CPe cells form polarised CPe vesicles when cultured in Matrigel, we have assessed their behaviour and potential use for pharmacological studies. Like CPe cells in vivo, CPe vesicles express transthyretin, E2f5, Fox-j1 and p73, and contain tight junctions, as indicated by ZO-1 expression and electron microscopy analysis. Time-lapse microscopy shows that CPe cells plated in Matrigel are highly migratory and rapidly form homotypic cell aggregates, which then reorganise to form vesicles whose size increases linearly overtime. Neither aggregate nor vesicle size is affected by AraC treatment, though this inhibitor significantly reduces proliferation in CPe monolayers. Increase in size of vesicles, which have reached a growth plateau is observed following addition of fluorescently-labelled CPe cells, which become incorporated into the vesicle walls. Significantly, treatment with secretion inhibitors blocks vesicle formation and their expansion. These results show that secretion, rather than cell division, controls vesicle growth, consistent with low levels of proliferation and thinning of the CPe observed both in growing vesicles and during CPe development. Therefore, changes in vesicle size can be used to evaluate the effect of putative molecules involved in the regulation of secretion.