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Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.

Trichinella spiralis is a parasitic nematode causing trichinellosis, a serious disease, and Caenorhabditis elegans is a free-living nematode, which is used as a model in parasitological studies. High levels of thymidylate synthase (EC 2.1.1.45; ThyA) and certain other enzymes involved in thymidylate biosynthesis were found throughout T. spiralis and C. elegans developmental cycles, including developmentally arrested forms, that is, T. spiralis muscle larva and C. elegans dauer larva. As ThyA activity is characteristic for cells that left the G0 phase of the cell cycle, an exceptional regulation of the cell cycle in nematodes is suggested, manifested by a global cell cycle arrest in developmentally arrested larvae of the two species. ThyA immunolocalization during development of T. spiralis and C. elegans revealed the presence of high enzyme levels not only in the developing embryos, where it was expected, but also in gonad primordia, egg and sperm cells, nerve ring and secretory cells, opening to T. spiralis esophagus and C. elegans pharynx, where it may point to those cell populations remaining cell cycle arrested.

Abstract: To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47phox subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47phox subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47phox subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host’s alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation. [Copyright &y& Elsevier]

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer. [ABSTRACT FROM AUTHOR]