DiS-C3(3) monitoring of in vivo mitochondrial membrane potential in C. elegans

The mitochondrial respiratory chain plays a crucial role in cellular and organismal health. In addition to being the major source of energy for most cells, mitochondrial respiratory chain function regulates or modulates redox and metabolite homeostasis, apoptosis and the generation of reactive oxygen species. In order to measure the relative in vivo mitochondrial membrane potential of different strains of the nematode, Caenorhabditis elegans, we have developed a fluorescence assay using the cationic, lipophilic carbocyanine dye, diS-C(3)(3). We demonstrate that two complex I-deficient mutants have significantly lower mitochondrial membrane potentials in vivo than wild type animals. Our fluorescence assay will enable us to better dissect and understand the complex phenotypic consequences of mitochondrial dysfunction.     

Protein disulfide isomerase activity is essential for viability and extracellular matrix formation in the nematode Caenorhabditis elegans

Protein disulfide isomerase (PDI) is a multifunctional protein required for many aspects of protein folding and transit through the endoplasmic reticulum. A conserved family of three PDIs has been functionally analysed using genetic mutants of the model organism Caenorhabditis elegans. PDI-1 and PDI-3 are individually non-essential, whereas PDI-2 is required for normal post-embryonic development. In combination, all three genes are synergistically essential for embryonic development in this nematode. Mutations in pdi-2 result in severe body morphology defects, uncoordinated movement, adult sterility, abnormal molting and aberrant collagen deposition. Many of these phenotypes are consistent with a role in collagen biogenesis and extracellular matrix formation. PDI-2 is required for the normal function of prolyl 4-hydroxylase, a key collagen-modifying enzyme. Site-directed mutagenesis indicates that the independent catalytic activity of PDI-2 may also perform an essential developmental function. PDI-2 therefore performs two critical roles during morphogenesis. The role of PDI-2 in collagen biogenesis can be restored following complementation of the mutant with human PDI.     

苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC50为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

Prolactin-like hormone in the nematode Trichinella spiralis larvae

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.     

Secreted virulence factors from Heterorhabditis bacteriophora highlight its utility as a model parasite among Clade V nematodes

Much of the available knowledge of entomopathogenic virulence factors has been gleaned from studies in the nematode parasite Steinernema carpocapsae, but there is good reason to complement this knowledge with similar studies in Heterorhabditis bacteriophora. Three candidate virulence factors from H. bacteriophora have recently been characterised, and each was demonstrated to contribute to infection. This information can be used not only to advance efforts in the biocontrol of insect pests, but also to make inferences about the emergence of parasitism among Clade V nematodes.     

Influence of ecological and edaphic factors on biodiversity of soil nematodes

Nematodes are the most diverse and highly significant group of soil-inhabiting microorganisms that play a vital role in organic material decomposition and nutrient recycling.Diverse geographical locations and environmental gradients show a significant impact on the diversity of nematodes. Present study aims to assess the effects of ecological (altitude, temperature, moisture) and edaphic (soil pH, nutrients, soil patches) factors on the soil nematode diversity and structure at five different landscape patches (forests, apple orchards, rice fields, pastures, and alpine zone) from ten different sites of Kashmir valley (India). Differences in the altitudinal gradients results in the shift of generic nematode population. Among the soil patches, highest nematode diversity was observed in forest soil and least in alpine soil; however, bacteriovorous nematodes dominated all the soil patches. The temperature and moisture have a significant effect on nematode diversity, the highest nematode trophic levels were observed above 21°C temperature, and 30% moisture. Nematode abundance decreased from alkaline to acidic pH of the soil. Soil nutrients such as, nitrogen (N) and phosphorus (P) have shown a detrimental effect in nematode richness at each site, where nematode diversity and richness of genera were higher at abundant soil N and P but decreased at low soil nutrients. Ecological indices like diversity index (DI), Shannon-Wiener Index (H'), enrichment index (EI), and maturity Index (MI) values demonstrated forest soil more favourable for nematodes and high soil health status than other soil patches. This study suggested that these indices may be helpful as soil monitoring tools and assessing ecosystem sustainability and biodiversity.     

Dissection of lin-11 enhancer regions in Caenorhabditis elegans and other nematodes

The Caenorhabditis elegans LIM homeobox gene lin-11 plays crucial roles in the morphogenesis of the reproductive system and differentiation of several neurons. The expression of lin-11 in different tissues is regulated by enhancer regions located upstream as well as within lin-11 introns. These regions are functionally separable suggesting that multiple regulatory inputs operate to control the spatiotemporal pattern of lin-11 expression. To further dissect apart the nature of lin-11 regulation we focused on three Caenorhabditis species C. briggsae, C. remanei, and C. brenneri that are substantially diverged from C. elegans but share almost identical vulval morphology. We show that, in these species, the 5′ region of lin-11 possesses conserved sequences to activate lin-11 expression in the reproductive system. Analysis of the in vivo role of these sequences in C. elegans has led to the identification of three functionally distinct enhancers for the vulva, VC neurons, and uterine π lineage cells. We found that the π enhancer is regulated by FOS homolog FOS-1 and LIN-12/Notch pathway effectors, LAG-1 (Su(H)/CBF1 family) and EGL-43 (EVI1 family). These results indicate that multiple factors cooperate to regulate π-specific expression of lin-11 and together with other findings suggest that the mechanism of lin-11 regulation by LIN-12/Notch signaling is evolutionarily conserved in Caenorhabditis species. Our work demonstrates that 4-way comparison is a powerful tool to study conserved mechanisms of gene regulation in C. elegans and other nematodes.     

Nippostrongylus brasiliensis: Infection decreases plasma butyrylcholinesterase activity in rats

Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.     

Characterisation of effector mechanisms at the host:parasite interface during the immune response to tissue-dwelling intestinal nematode parasites

The protective immune response that develops following infection with many tissue-dwelling intestinal nematode parasites is characterised by elevations in IL-4 and IL-13 and increased numbers of CD4+ T cells, granulocytes and macrophages. These cells accumulate at the site of infection and in many cases can mediate resistance to these large multicellular pathogens. Recent studies suggest novel potential mechanisms mediated by these immune cell populations through their differential activation and ability to stimulate production of novel effector molecules. These newly discovered protective mechanisms may provide novel strategies to develop immunotherapies and vaccines against this group of pathogens. In this review, we will examine recent studies elucidating mechanisms of host protection against three widely-used experimental murine models of tissue-dwelling intestinal nematode parasites: Heligmosomoides polygyrus, Trichuris muris and Trichinella spiralis.