Transcriptomic and Coexpression Network Analyses Revealed Pine Chalcone Synthase Genes Associated with Pine Wood Nematode Infection.

Pine wood nematode (PWN) causes serious diseases in conifers, especially pine species. To investigate the transcriptomic profiles of genes involved in pine-PWN interactions, two different pine species, namely, Pinus thunbergii and P. massoniana, were selected for this study. Weighted gene coexpression network analysis (WGCNA) was used to determine the relationship between changes in gene expression and the PWN population after PWN infection. PWN infection negatively affects the expression of most genes in pine trees, including plant defense-related genes such as genes related to plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway in plants. However, the expression of chalcone synthase genes and their related genes were proportional to the changes in nematode populations, and chalcone synthase genes were dominant within the coexpression module enriched by genes highly correlated with the nematode population. Many genes that were closely related to chalcone synthase genes in the module were related to flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Pine trees could actively adjust their defense strategies in response to changes in the number of invasive PWNs, but the sustained expression of chalcone synthase genes should play an important role in the inhibition of PWN infection.

Cloning and characterisation of a Heterodera glycines aminopeptidase cDNA.

An aminopeptidase full-length cDNA (Hg-amp-1) was cloned from the adult female soybean cyst nematode Heterodera glycines by heterologous screening of a cDNA library with a Caenorhabditis elegans EST sequence. The predicted open reading frame encoded an 882-amino acid protein containing the conserved zinc-binding domain and GAMEN motif that are characteristic of M1 family aminopeptidases. The putative protein lacks any subcellular targeting signals and displays strong similarity to puromycin-sensitive aminopeptidases from C. elegans, Drosophila and mammals. Hg-amp-1 is expressed in juvenile nematodes and both male and female adults, with highest expression in gravid females. In situ mRNA hybridisation localised the Hg-amp-1 transcript to the genital primordium of pre-parasitic juvenile nematodes and the reproductive tract of adult females. Suppression of Hg-amp-1 transcript level by RNA-interference led to a 61% reduction in the number of female nematodes parasitising soybean roots 21 days post infection with infective juvenile nematodes that had been exposed to double-stranded RNA.

Identification of the secreted neutral proteases from Anisakis simplex.

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.

Suppression of Nippostrongylus brasiliensis (Nematoda)-induced lysophospholipase activity and peripheral eosinophilia by Eimeria nieschulzi (Apicomplexa).

Interspecific interactions between Nippostrongylus brasiliensis and Eimeria nieschulzi were studied by measuring fecal lysophospholipase (LYPH) activity and relative numbers of peripheral eosinophils in rats singly or concurrently infected with one or both parasite species. Three groups of 10 rats each were inoculated with 2 X 10(3) N. brasiliensis L3 larvae and/or 5 X 10(5) E. nieschulzi sporulated oocysts. Groups 1 and 2 were infected with E. nieschulzi or N. brasiliensis, respectively. Group 3 rats were infected first with N. brasiliensis, followed on day 8 postinoculation (PI) with E. nieschulzi. Each rat served as its own control. Results revealed LYPH levels rose steadily in Group 2 rats, reaching significant peaks on days 10 and 12 PI before decreasing to control levels. Lysophospholipase activity in Groups 1 and 3, however, did not differ from control values. Group 2 rats also demonstrated peripheral eosinophilia, with peak values occurring on days 10, 12, 14, and 16 PI, while rats in Groups 1 and 3 exhibited no eosinophilia. These results demonstrate that E. nieschulzi suppressed intestinal LYPH activity and relative peripheral eosinophilia and demonstrate that a host's immune response to a single parasite may be significantly altered when a second parasite species is present.

Apicomplexa, trypanosoma and parasitic nematode protein kinases as antiparasitic therapeutic targets.

Parasitic infections caused by Plasmodium, Trypanosoma, Leishmania, Toxoplasma and parasitic nematodes affect hundreds of millions of individuals worldwide and are the cause of significant mortality and morbidity, particularly in developing countries. These diseases also have an impact on individuals from developed countries; for example, some US troops in Iraq and Afghanistan have been infected with Leishmania. The annual mortality associated with parasitic infections is estimated to be 1.5 million deaths. The socioeconomic impact of the morbidity associated with parasitic infections is significant, and the development of new drugs, aimed at novel targets, is urgently needed to develop effective treatments for these diseases. The small-molecule inhibitors discussed in this review constitute useful tools with which to explore the relevance of kinase inhibition in inducing antiparasitic activity. The aim of recent target-based approaches used in the development of parasite kinase inhibitors is to identify novel antiparasitic agents with therapeutic potential.

Fluctuations in rat liver alanine-amino-transferase activity during experimental nippostrongylosis.

The activity of the gluconeogenic enzyme, alanine-amino-transferase (ALT), in a preparation from the liver of rats was studied by means of an in vitro assay throughout the course of a primary infection of Nippostrongylus brasiliensis, established by a subcutaneous injection of approximately 4000 3rd-stage larvae. The activity was measured on days 1-14 p.i. in both uninfected and infected rats and a marked pattern in the enzyme's activity was observed. In infected rats, the activity increased from 1.46 +/- 0.19 U/g liver on day 1 p.i. to a peak on day 4 p.i. of 10.75 +/- 1.62 U/g liver, then decreased to a trough of 0.44 +/- 0.18 U/g liver on day 10 p.i. before returning to original levels by day 14 p.i., by which time the infection had been largely eliminated. In uninfected rats the activity of the liver enzyme remained constant throughout this period with a value of 2.54 +/- 0.12 U/g liver. The activity of the enzyme in vitro was found to be related to the size of the inoculum on days 4 and 10 p.i. It was proposed that these observations could be due to either (1) a direct effect of the parasite, or (2) a consequence of the host immune response to the infection. In order to investigate the second proposition more fully, liver ALT activity was investigated by in vitro assay on selected days p.i. in rats experiencing a secondary N. brasiliensis infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase B in the brains and meninges of nonsensitized and sensitized rats after challenge with Angiostrongylus cantonensis.

After a primary infection with 100 Angiostrongylus cantonensis larvae, infected rats showed elevated phospholipase B activity in meningeal and brain homogenates beginning with the first week and continuing through the first month of infection. The rise in phospholipase B values through the first 4 weeks, with a prolonged peak spanning days 30 to 31, coincided with the invasion and maturation of the parasites in the brain, and the ensuing sharp decline in phospholipase B levels, shown by the readings on day 45, coincided in turn with the known migration of the worms from the brain to the lungs, which begins about 5 weeks after infection. In the meninges, the pattern of enzyme elevation was generally similar to that in the brain samples except that the highest activity was seen earlier at days 8 to 9, followed by a gradual decline by days 30 to 31 and a sharper drop by day 45. Rats challenged with 100 larvae 53 days after the primary infection exhibited an almost immediate rise of phospholipase B activity in both the brain and meninges; the peaks of activity occurred at day 1 for the meninges and day 25 for the brain, and levels above control values were still present at day 50. Comparison of the total enzymatic content of the cerebral tissue and meninges revealed that a remarkably high proportion of the phospholipase B activity was contained in the meninges. The inference that elevated levels of this enzyme in the cerebral tissue of A. cantonensis-infected rats are due to inflammatory reactions within the meningeal envelopes was confirmed by histochemical demonstration of specific sites of enzymatic activity limited to the meninges. It is of interest that 80% of the cells positive for the enzyme were clearly identifiable as eosinophils since an association of bone marrow eosinophilia and high phospholipase B levels in rats infected with A. cantonensis was shown in our earlier study of rats infected with this parasite.

Testing the hypothesis that crypt cell hyperplasia inhibits lactase expression by mouse jejunal enterocytes.

Lactase activity and crypt cell proliferation both increased significantly in mouse jejunal villi in the presence of the intestinal parasite Nematospiroides dubius. Comparisons are made between this result and others showing lactase activity to decline whenever crypt cell proliferation is increased.

Intestinal infection with Nematospiroides dubius selectively increases lactase expression by mouse jejunal enterocytes.

1. Intestinal structure, lactase (beta-galactosidase; EC 3.2.1.23) activity and alkaline phosphatase activity have been determined in mouse jejunal and ileal tissues before and during infection with the intestinal parasite Nematospiroides dubius. 2. Oral infection with small numbers of N. dubius larvae caused villus height, crypt depth and enterocyte migration rate to increase in the mouse jejunum. None of these effects occurred in ileal tissue. 3. Lactase activity also increased in jejunal, but not ileal, tissue of infected mice. This increase was associated with a doubling of the rate at which activity appeared in the brush-border membrane of enterocytes during migration over the basal regions of jejunal villi. Alkaline phosphatase activity in jejunal tissue remained unchanged in infected mice. 4. Attention is drawn to the fact that this is the first occasion when crypt cell hyperplasia has been found to be positively correlated with an increase in lactase activity and a decrease in cytotoxic/suppressor T-cells. Further work is needed to establish the primary cause of these effects.