Characterization of vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase in osteoclasts

V-ATPase plays important roles in controlling the extra- and intra-cellular pH in eukaryotic cell, which is most crucial for cellular processes. V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and integral V(0) domain responsible for proton translocation. Osteoclasts are multinucleated cells responsible for bone resorption and relate to many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone loss. This review summarizes the structure and function of V-ATPase and its subunit, the role of V-ATPase subunits in osteoclast function, V-ATPase inhibitors for osteoclast function, and highlights the importance of V-ATPase as a potential prime target for anti-resorptive agents.     

Nematode sperm

Parasitic nematode infections remain a major public health problem in many parts of the world. Because most of the current strategies aimed at controlling parasitic nematode infections have met with only limited success, it may be time to consider alternative approaches. An aspect of nematode biology that has drawn little attention as a target for control is the reproductive process. Although there are numerous facets of the overall reproductive biology of nematodes that hold potential as targets for intervention, Alan Scott here focuses on the male reproductive system, and outlines some of the known unique processes and characteristics of sperm formation and sperm function that could be exploited to block fertilization.     

The diversity of ABC transporter genes across the Phylum Nematoda

It is estimated that one billion people globally are infected by parasitic nematodes, with children, pregnant women, and the elderly particularly susceptible to morbidity from infection. Control methods are limited to de-worming, which is hampered by rapid re-infection and the inevitable development of anthelmintic resistance. One family of proteins that has been implicated in nematode anthelmintic resistance are the ATP binding cassette (ABC) transporters. ABC transporters are characterized by a highly conserved ATP-binding domain and variable transmembrane regions. A growing number of studies have associated ABC transporters in anthelmintic resistance through a protective mechanism of drug efflux. Genetic deletion of P glycoprotein type ABC transporters in Caenorhabditis elegans demonstrated increased sensitivity to anthelmintics, while in the livestock parasite, Haemonchus contortus, anthelmintic use has been shown to increase the expression of ATP transporter genes. These studies as well as others, provide evidence for a potential role of ABC transporters in drug resistance in nematodes. In order to understand more about the family of ABC transporters, we used hidden Markov models to predict ABC transporter proteins from 108 species across the phylum Nematoda and use these data to analyze patterns of diversification and loss in diverse nematode species. We also examined temporal patterns of expression for the ABC transporter family within the filarial nematode Brugia malayi and identify cases of differential expression across diverse life-cycle stages. Taken together, our data provide a comprehensive overview of ABC transporters in diverse nematode species and identify examples of gene loss and diversification in nematodes based on lifestyle and taxonomy.     

Cloning and biochemical characterization of blisterase,a subtilisin-like convertase from the filarial parasite,Onchocerca volvulus

Blisterase is a subtilisin-like proprotein convertase of nematodes. The enzyme is named after the blistered cuticle found in Caenorhabditis elegans with the bli-4 e937 mutation. The critical role of the enzyme in cuticle production makes it a potential drug target for parasitic nematodes. We have cloned and expressed blisterase from the parasitic nematode Onchocerca volvulus, a major cause of blindness in Africa. The catalytic domain of the protease exhibits 84% identity with the corresponding domain of its closest homologue, C. elegans blisterase. O. volvulus blisterase expressed in insect cells has maximal activity in 1 mm calcium at neutral pH. The protease is inhibited by EDTA, the suicide substrate decanoyl-RVKR-chloromethylketone, alpha1-antitrypsin Portland and by its own propeptide. Substrate assays with fluorescent peptides show that O. volvulus blisterase requires a P4 arginine and a basic amino acid at P1 for cleavage. The kcat of blisterase on the peptide substrate, t-butyloxycarbonyl-RVRR-4-methylcoumaryl-7-amide was determined to be 0.018 s-1. In vitro cleavage studies with the nematode polyprotein antigen demonstrated that blisterase cleaved at tetrabasic (RRKR) but not at dibasic (KR) sites. This report describes the first biochemical characterization of the nematode specific protease, blisterase.     

Parasitism of the mosquito Culex pipiens by the nematode Heterorhabditis bacteriophora

Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culex pipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts' defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed.     

Influence of Nematodes on Resource Utilization by Bacteria—an in vitro Study

The positive influence of bacterial feeding nematodes on bacterial mediated processes such as organic matter mineralization and nutrient cycling is widely accepted, but the mechanisms of these interactions are not always apparent. Both transport of bacteria by nematodes, and nutritional effects caused by nematode N excretion are thought to be involved, but their relative importance is not known because of the difficulties in studying these interactions in soil. We developed a simple in vitro assay to study complex nematode/bacterial interactions and used it to conduct a series of experiments to determine the potential influence of nematode movement and nutritional effects on bacterial resource use. The system used bacterial feeding and nonfeeding insect parasitic nematodes, and luminescent bacteria marked with metabolic reporter genes. Both nutritional enhancement of bacterial activity and bacterial transport were observed and we hypothesize that in nature, the relative importance of transport is likely to be greater in bulk soil, whereas nematode excretion may have greater impact in the rhizosphere. In both cases, the ability of nematodes to enhance bacterial resource utilization has implications for soil components of biogeochemical cycling.     

Thermoregulation in the life cycle of nematodes

An unanswered question in the biology of many parasites is the mechanism by which environmental (or external) and intrinsic signals are integrated to determine the switch from one developmental stage to the next. This is particularly pertinent for nematode parasites, many of which have a free-living stage in the environment prior to infection of the mammalian host, or for parasites such as filarial nematodes, which utilise an insect vector for transmission. The environmental changes experienced by a parasite upon infection of a mammalian host are extremely complex and poorly understood. However, the ability of a parasite to sense its new environment must be intrinsically linked to its developmental programme, as progression of the life cycle is dependent upon the infection event. In this review, the relationship between temperature and development in filarial nematodes and in the free-living species Caenorhabditis elegans is summarised, with a focus on the role of heat shock factor and heat shock protein 90 in the nematode life cycle.     

Utility of nematode community analysis as an integrated measure of the functional state of soils: perspectives and challenges

Soil nematode communities have the potential to provide unique insights into many aspects of soil processes. Since most nematodes are active in soil throughout the year, they can potentially provide a holistic measure of the biotic and functional status of soils. In contrast to other soil microbial groups, representative samples of soil nematode communities are relatively easy to obtain. However, most current nematode ecological information has been survey-based or purely observational in nature, with a persistent focus on detailed taxonomic analysis of nematode communities. The development of a Maturity Index, MI, represents a significant advance in classifying communities and it continues to be refined and developed. But, to develop a wide capacity to use soil nematode information for diagnostic and predictive purposes, particularly for agricultural soils, we need a new, more robust approach, which does not require extensive taxonomic skill and includes more functional criteria. One of the key attributes of nematodes is the relationship between structural form (principally oesophagal feeding apparatus) and function (i.e. trophic group). Nematode form is readily determinable by direct observation of extracted nematodes and high-level taxonomic skills are not needed to assign the major community components to their different trophic and ecological groups. Consequently, the trophic structure of nematode communities is relatively easy to determine and can provide an integrated measure of the status of the other groups on which they feed. Similarly, population numbers and proportions of juveniles and adults can be readily determined, permitting calculation of relative biomass and dynamics of population growth. The size distribution of individuals within the community is likely also to be an indicator of the structural status of soils from a biotic standpoint. However, fundamental gaps remain in our understanding which limit our ability to relate differences in nematode communities to functional differences. There needs to be a greater emphasis on the development and experimental testing of hypotheses, a greater integration of nematology into soil-process related studies, and the development of a specific, soil-nematode related theoretical framework for understanding epidemiological and soil colonisation processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant-nematode interactions

Root-knot nematodes and cyst nematodes are obligate, biotrophic pathogens of numerous plant species. These organisms cause dramatic changes in the morphology and physiology of their hosts. The molecular characterization of induced plant genes has provided insight into the plant processes that are usurped by nematodes as they establish their specialized feeding cells. Recently, several gene products have been identified that are secreted by the nematode during parasitism. The corresponding genes have strong similarity to microbial genes or to genes that are found in nematodes that parasitize animals. New information on host resistance genes and nematode virulence genes provides additional insight into this complex interaction.     

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.     

Vacuolar-type proton ATPase as regulator of membrane dynamics in multicellular organisms

Acidification inside membrane compartments is a common feature of all eukaryotic cells. The acidic milieu is involved in many physiological processes including secretion, protein processing, and others. However, its cellular relevance has not been well established beyond the results of in vitro studies involving cultured cell systems. In the last decade, human and mouse genetics have revealed that the acidification machinery is implicated in multiple pathophysiological disorders, and thus our understanding of physiological consequences of the defective acidification in multicellular organisms has improved. In invertebrates including Drosophila and nematodes, mutations of V-ATPase were found to lead the development of rather unexpected phenotypes. Studies have suggested that V-ATPase may be involved in membrane fusion and vesicle formation, important processes for membrane trafficking, and have further implied its involvement in cell–cell fusion. This rather novel idea arose from the phenotypes associated with genetic disorders involving V-ATPase genes in various genetic model systems. In this article, we focus and overview the non-classical, beyond proton-pumping function of the vacuolar-type ATPase in exo/endocytic systems.     

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.     

Nicotinic acetylcholine receptors: A comparison of the nAChRs of Caenorhabditis elegans and parasitic nematodes

Nicotinic acetylcholine receptors (nAChRs) play a key role in the normal physiology of nematodes and provide an established target site for anthelmintics. The free-living nematode, Caenorhabditis elegans, has a large number of nAChR subunit genes in its genome and so provides an experimental model for testing novel anthelmintics which act at these sites. However, many parasitic nematodes lack specific genes present in C. elegans, and so care is required in extrapolating from studies using C. elegans to the situation in other nematodes. In this review the properties of C. elegans nAChRs are reviewed and compared to those of parasitic nematodes. This forms the basis for a discussion of the possible subunit composition of nAChRs from different species of parasitic nematodes. Currently our knowledge on this is largely based on studies using heterologous expression and pharmacological analysis of receptor subunits in Xenopus laevis oocytes. It is concluded that more information is required regarding the subunit composition and pharmacology of endogenous nAChRs in parasitic nematodes.     

The worm has turned--microbial virulence modeled in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a facile and economical model host for the study of evolutionarily conserved mechanisms of microbial pathogenesis and innate immunity. A rapidly growing number of human and animal microbial pathogens have been shown to injure and kill nematodes. In many cases, microbial genes known to be important for full virulence in mammalian models have been shown to be similarly required for maximum pathogenicity in nematodes. C. elegans has been used in mutation-based screening systems to identify novel virulence-related microbial genes and immune-related host genes, many of which have been validated in mammalian models of disease. C. elegans-based pathogenesis systems hold the potential to simultaneously explore the molecular genetic determinants of both pathogen virulence and host defense.     

Detection and Description of Soils with Specific Nematode Suppressiveness

Soils with specific suppressiveness to plant-parasitic nematodes are of interest to define the mechanisms that regulate population density. Suppressive soils prevent nematodes from establishing and from causing disease, and they diminish disease severity after initial nematode damage in continuous culturing of a host. A range of non-specific and specific soil treatments, followed by infestation with a target nematode, have been employed to identify nematode-suppressive soils. Biocidal treatments, soil transfer tests, and baiting approaches together with observations of the plant-parasitic nematode in the root zone of susceptible host plants have improved the understanding of nematode-suppressive soils. Techniques to demonstrate specific soil suppressiveness against plant-parasitic nematodes are compared in this review. The overlap of studies on soil suppressiveness with recent advances in soil health and quality is briefly discussed. The emphasis is on methods (or criteria) used to detect and identify soils that maintain specific soil suppressiveness to plant-parasitic nematodes. While biocidal treatments can detect general and specific soil suppressiveness, soil transfer studies, by definition, apply only to specific soil suppressiveness. Finally, potential strategies to exploit suppressive soils are presented.     

Research collaborations can improve the use of organic amendments for plant-parasitic nematode management

The concept of utilizing organic amendments to manage plant-parasitic nematodes is not new, but the widespread implementation of this management practice has still not been realized. The use of organic amendments for plant-parasitic nematode management is a complex process requiring an understanding of the transformation and generation of active compounds. As a result, research endeavors to understand and maximize the use of this management practice require a multidisciplinary approach which draws upon the expertise of nematologists, microbiologists, natural product chemists and soil scientists. Factors that require analysis and clarification include lethal concentration levels of organic amendments necessary to kill nematodes; chemical composition of incorporation material; fate and exposure potential to nematodes of compounds released into the soil; and the influence of environmental factors (i.e., temperature, microbial community, soil type) on the activity of organic amendments. Examples of research conducted in a collaborative manner with rye (Secale cereale) and a biosolid amendment demonstrate the power of multidisciplinary research. Only through collaborative research can consistent and reliable nematode suppression with organic amendments be achieved.     

Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics

Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.     

The Surface Coat of Plant-Parasitic Nematodes: Chemical Composition,Origin, and Biological Role—A Review

Chemical composition, origin, and biological role of the surface coat (SC) of plant-parasitic nematodes are described and compared with those of animal-parasitic and free-living nematodes. The SC of the plant-parasitic nematodes is 5-30 nm thick and is characterized by a net negative charge. It consists, at least in part, of glycoproteins and proteins with various molecular weights, depending upon the nematode species. The lability of its components and the binding of human red blood cells to the surface of many tylenchid plant-parasitic nematodes, as well as the binding of several neoglycoproteins to the root-knot nematode Meloidogyne, suggest the presence of carbohydrate-recognition-domains for host plants and parasitic or predatory soil microorganisms (Pasteuria penetrans and Dactylaria spp., for example). These features may also assist in nematode adaptations to soil environments and to plant hosts with defense mechanisms that depend on reactions to nematode surfaces. Surface coat proteins can be species and race specific, a characteristic with promising diagnostic potential.