Search

All species in the genus Trichinella, between them T. spiralis and T. pseudospiralis, have been successful in colonizing striated sceletal muscle tissue and remain infective in this niche for months to years. Trichinella spiralis causes trichinellosis, a serious disease in man and other mammals. Mating of adult worms (developing from infective larvae, deriving from digested infected meat) occurs in a non membrane-bound portion of columnar epithelium of the host΄s small intestine. The fertilized females enter the intestinal wall and release to the bloodstream the newborn larvae. Each of these penetrates host΄s skeletal muscle cell and lives in its modified portion, the nurse cell, surrounded by a collagen capsule around which a circulatory rete develops. The nurse cell development, initiated by T. spiralis infection, is associated with a variety of changes, including cell cycle re-entry and induction of DNA synthesis, followed by the apparent G2/M arrest of the infected cell in the cell cycle. Similar changes appear to be caused by T. pseudospiralis infection, albeit the nurse cell complexes are not encapsulated by collageneous fibres and the larvae may move between muscle cells. Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2'-deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of N5,10-methylenetetrahydrofolate, with concomitant production of thymidylate and dihydrofolate. The coenzyme tetrahydrofolate is regenerated via dihydrofolate reduction by the enzyme dihydrofolate reductase (DHFR). One of the sources of TS substrate, dUMP, is dUTP hydrolysis in a pyrophosphatase reaction catalyzed by the enzyme dUTPase. TS and dUTPase induction is known to be associated with cell proliferation. Thymidylate synthesis inhibition by drugs targeted at either TS or DHFR is taken advantage of in chemotherapy. TS, DHFR and dUTPase were found to be persistently expressed at a high and constant level, comparable to that found in regenerating rat liver, in crude extracts from adult worms of Trichinella spiralis, as well as from developmentally arrested muscle larvae of both Trichinella spiralis (isolated 1–24 months after infection) and Trichinella pseudospiralis (isolated 5.5–13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. Moreover, T. spiralis muscle larvae (T. pseudospiralis larvae were not tested) contained also high level, comparable with that found in mouse leukemia L1210 cells, of DNA polymerase α, a key enzyme of the eukaryotic replication complex, its expression also known to be associated with cell proliferation. Immunofluorescent detection of TS protein was done with the use of monoclonal antibodies, developed by in vivo immunization of Balb/c mice with homogeneous recombinant rat hepatoma TS protein as an antigen. The specific anti-rat TS antibodies recognized also T. spiralis TS, as indicated by cross-reactivity on Western blot. Localization of the enzyme was based on analysis of pictures collected by confocal microscopy. Two types of T. spiralis muscle larvae preparations were studied: muscle larvae isolated from mouse muscles by a procedure destroying nurse cells and muscle larvae remaining in nurse cells, isolated as an intact nurse cell preparation. The results revealed reproducible TS localization patterns, reflected by strong fluorescence emitted by cells of both female and male gonad primordium, as well as from the regions around stichocyte nuclei. [ABSTRACT FROM AUTHOR]