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Myosin A, an unconventional class XIV myosin of the protozoan parasite Toxoplasma gondii, undergoes calcium-dependent phosphorylation, providing a mechanism by which the parasite can regulate motility-based processes such as escape from the infected host cell at the end of the parasite's lytic cycle.

ABSTRACTThe apicomplexan parasites Cryptosporidium parvumand Cryptosporidium hominisare major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidiumbiology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvumgrowth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondiiand C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvumand other apicomplexan parasites.

The article focuses on the importance of government contracting principles in the wake of the budget uncertainty in the federal government in the U.S. Topic include the attempt of the government to stretch its budget by exercising its inspection and warranty rights, the need for contractors to meet the demand of the government for more performance out of its existing contracts, and the parties' rights and obligations under various warranty clauses.

The article focuses on the involvement of the U.S. military troops on Grenada, an island on Caribbean Sea, to prevent the political turmoil. It states that the seizure of the Grenadian government by the Marxist People's Revolutionary Army (PRA) led by General Hudson Austin and Deputy Prime Minister Bernard Coard led to the interference of the U.S. military. It mentions that the political conflict occurred after the Marxists executed Prime Minister Maurice Bishop on October 19, 1983.

The article focuses on the encounter between the 1st Infantry Division of the U.S. Army and the 5th Division of South Vietnam at the Loc Ninh Rubber Plantation in Vietnam on June 11, 1966. Topics discussed include the statement of Richard G. Kurtz, a retired Army Colonel, on the importance and singularity of the event, the description of the battlefield, and the comment of former Lieutenant Colonel Kyle W. Bowie on the role of the rubber trees during the battle.

ABSTRACTToxoplasma gondiimotility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser163and Ser167. The importance of these phosphorylation events was determined by mutation of Ser163and Ser167to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser163and Ser167to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser163and Ser167to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser163and Ser167to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser163and Ser167in GAP45 controls the final step in assembly of the myosin XIV motor complex.

ABSTRACTLimited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondiiaccompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasmatachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[125I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [125I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

ABSTRACTThe pellicle of the protozoan parasite Toxoplasma gondiiis a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[125I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [125I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T.gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T.gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion.We present here Laser Scanning Cytometer (LSC)‐based assays of T.gondii invasion and attachment. The invasion assay involves automated counting of invaded and non‐invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment.The LSC‐based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope‐based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms.The LSC‐based assays represent useful new methods for quantitatively measuring attachment and invasion by T.gondii, and can be readily adapted to study similar processes in other host–pathogen systems. © 2005 Wiley‐Liss, Inc.

Toxoplasma gondiiis an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondiior related apicomplexan parasites (including Plasmodiumspp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1(TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondiitachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.

ABSTRACTMany intracellular pathogens are separated from the cytosol of their host cells by a vacuole membrane. This membrane serves as a critical interface between the pathogen and the host cell, across which nutrients are imported, wastes are excreted, and communication between the two cells takes place. Very little is known about the vacuole membrane proteins mediating these processes in any host-pathogen interaction. During a screen for monoclonal antibodies against novel surface or secreted proteins of Toxoplasma gondii, we identified ROP4, a previously uncharacterized member of the ROP2 family of proteins. We report here on the sequence, posttranslational processing, and subcellular localization of ROP4, a type I transmembrane protein. Mature, processed ROP4 is localized to the rhoptries, secretory organelles at the apical end of the parasite, and is secreted from the parasite during host cell invasion. Released ROP4 associates with the vacuole membrane and becomes phosphorylated in the infected cell. Similar results are seen with ROP2. Further analysis of ROP4 showed it to be phosphorylated on multiple sites, a subset of which result from the action of either host cell protein kinase(s) or parasite kinase(s) activated by host cell factors. The localization and posttranslational modification of ROP4 and other members of the ROP2 family of proteins within the infected cell make them well situated to play important roles in vacuole membrane function.

ABSTRACTAs is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of Toxoplasma gondiitachyzoites. The mechanisms by which T. gondiiGPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown. As a first step in developing tools to study these processes, we show here that Clostridium septicumalpha-toxin, a pore-forming toxin that targets GPI-anchored protein receptors on the surface of mammalian cells, is active against T. gondiitachyzoites (50% effective concentration, 0.2 nM). Ultrastructural studies reveal that a tight physical connection between the plasma membrane and the underlying membranes of the inner membrane complex is locally disrupted by toxin treatment, resulting in a massive outward extension of the plasma membrane and ultimately lysis of the parasite. Toxin treatment also causes swelling of the parasite endoplasmic reticulum, providing the first direct evidence that alpha-toxin is a vacuolating toxin. Alpha-toxin binds to several parasite GPI-anchored proteins, including surface antigen 3 (SAG3) and SAG1. Interestingly, differences in the toxin-binding profiles between the virulent RH and avirulent P strain were observed. Alpha-toxin may prove to be a powerful experimental tool for molecular genetic analysis of GPI anchor biosynthesis and GPI-anchored protein trafficking in T. gondiiand other susceptible protozoa.

The article presents a description of Vietnam War veterans including Second Lieutenant Stephen Douglas, Sergeant Oliver J. Fugere, Jr and Sergeant First Class Juan Santiago of the fight at Michelin Rubber Plantation at Ap Nha Mat in Vietnam against the Dong Xoai Regiment. It mentions the valor of the soldiers and the confusion that arose during the shooting which resulted in loss of 50 percent of the U.S. soldiers. It also mentions the memories of survivors of the friends lost in the shooting.

The article offers information on Americans fought with the French Foreign Legion (FFL), a military service wing of the French Army during World War 1914-1918. The topics discussed include brief information on John Bowe, a veteran of the Philippines War, who earned the Serbian War Cross, Edgar Bouligny, who was described as the best boxer and runner in his company, and medals won by American legionnaires.

Several letters to the editor are presented in response to articles in the previous issues which include one on employment issues, one about the theistic evolution, and one about secular science.

The article reflects on a battle fought between the U.S. Army and its Philippine allies with the Muslims of Philippines in Mindanao Islands, Philippines from June 11-15, 1913. It mentions about U.S. army officials John J. Pershing and James L. Collins who reflect on their experiences regarding the battle. It states that the battle was started because Moros have refused to surrender in front of the U.S. Army and as a result 500 Moros were decimated.

In this article, the authors' outlines several factors which contribute for success of physical fitness club. Topics discussed include completion of customer loyalty survey by the customer management software service provider Retention People and Great Britain-based researchers Leisure-Net Solutions Ltd., promoters' trends in top performing clubs and Net Promoter Score for the customers.

ABSTRACTApical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondiithat plays a critical role in host cell invasion. The ligand to which T. gondiiAMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.IMPORTANCENearly one-third of the world’s population is infected with the protozoan parasite Toxoplasma gondii, which causes life-threatening disease in neonates and immunocompromised individuals. T. gondiiis a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

Several letters to the editor in response to articles in previous issues including "A Fashionable Life" in the September 2006 issue, "God Bless the Child," by Laverne Ballard in the September issue and "Like a Virgin," by Derek Westbrook in the September issue.

Discusses the use of color for job stress management. Effect of color spectrum on personality; Violet for conflict avoidance; Blue for relaxation; Replacement of green with pink and orange; Red and yellow for stimulation; Color combinations.

The article mentions the 75th anniversary of Robert M. Losey, U.S. Army Air Forces Captain, who was also the first American military casualty in World War II.

Several letters to the editor are presented in response to articles in previous issues including "Excellence Marks the Spots" by Lonna Tucker in the August 2013 issue, "He Still Calls Them 'Anasazi'" by David Roberts in the August 2013 issue, and "Hike of the Month" in the April 2013 issue.

The article explores the application of the alternating current permanent-magnet (ACPM) on elevators. The drives used by majority of main manufacturers have the capability of operating ACPM machines. The permanent magnets used for ACPM technology are described as rare earth magnets composed of iron oxide, bromine and other metal oxides. Neodymium ceramic magnets are utilized for gearless AC synchronous elevator machineries.