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Developmental arrest in Caenorhabditis elegans dauer larvae causes high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae

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Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.
... In our previous studies, we were interested in Trichinella spiralis, which is responsible in both developing and developed countries for a serious disease, i.e., trichinellosis [13], and a free-living nematode Caenorhabditis elegans, which is often used as a model organism in parasitological studies [14][15][16]. Of particular interest was a high TS-specific activity present throughout the developmental cycles of the two nematode species, including their developmentally arrested forms (lacking cell proliferation and thus expected to show TS activity either low or none at all), including T. spiralis infective muscle larvae [16][17][18][19] and C. elegans dauer larvae [17], the latter corresponding to developmentally arrested infective larvae of parasitic nematodes [14]. It pointed to the high TS level as a result of an unusual cell cycle regulation, leading to a long-term cell cycle arrest, in the developmentally arrested larvae (discussed in Reference [17,18]). ...
... In our previous studies, we were interested in Trichinella spiralis, which is responsible in both developing and developed countries for a serious disease, i.e., trichinellosis [13], and a free-living nematode Caenorhabditis elegans, which is often used as a model organism in parasitological studies [14][15][16]. Of particular interest was a high TS-specific activity present throughout the developmental cycles of the two nematode species, including their developmentally arrested forms (lacking cell proliferation and thus expected to show TS activity either low or none at all), including T. spiralis infective muscle larvae [16][17][18][19] and C. elegans dauer larvae [17], the latter corresponding to developmentally arrested infective larvae of parasitic nematodes [14]. It pointed to the high TS level as a result of an unusual cell cycle regulation, leading to a long-term cell cycle arrest, in the developmentally arrested larvae (discussed in Reference [17,18]). ...
... Of particular interest was a high TS-specific activity present throughout the developmental cycles of the two nematode species, including their developmentally arrested forms (lacking cell proliferation and thus expected to show TS activity either low or none at all), including T. spiralis infective muscle larvae [16][17][18][19] and C. elegans dauer larvae [17], the latter corresponding to developmentally arrested infective larvae of parasitic nematodes [14]. It pointed to the high TS level as a result of an unusual cell cycle regulation, leading to a long-term cell cycle arrest, in the developmentally arrested larvae (discussed in Reference [17,18]). Although TS protein in those larvae is probably catalytically irrelevant (no DNA synthesis), it may play a regulatory role in view of the enzyme's certain non-catalytic activities, including capacity to bind mRNA (its own and some others) and inhibit translation, with potential regulation of several cellular genes [19,20], as well as an oncogene-like activity [21]. ...
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With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.
... All rights reserved. opmentally arrested muscle larvae [16][17][18][19][20]. Notably, we found similar phenomenon to take place in the development of the free living nematode Caenorhabditis elegans [18], whose developmentally arrested dauer larvae correspond to developmentally arrested infective larvae of parasitic nematodes [6], such as T. spiralis muscle larvae. ...
... All rights reserved. opmentally arrested muscle larvae [16][17][18][19][20]. Notably, we found similar phenomenon to take place in the development of the free living nematode Caenorhabditis elegans [18], whose developmentally arrested dauer larvae correspond to developmentally arrested infective larvae of parasitic nematodes [6], such as T. spiralis muscle larvae. An unusual cell cycle regulation, involving long term cell cycle arrest, is suggested to play a role in developmentally arrested Trichinella muscle larvae and C. elegans dauer larvae, resulting, among others, in high TS level (discussed in [16,18]). ...
... opmentally arrested muscle larvae [16][17][18][19][20]. Notably, we found similar phenomenon to take place in the development of the free living nematode Caenorhabditis elegans [18], whose developmentally arrested dauer larvae correspond to developmentally arrested infective larvae of parasitic nematodes [6], such as T. spiralis muscle larvae. An unusual cell cycle regulation, involving long term cell cycle arrest, is suggested to play a role in developmentally arrested Trichinella muscle larvae and C. elegans dauer larvae, resulting, among others, in high TS level (discussed in [16,18]). TS protein, present in developmentally arrested forms, is probably catalytically irrelevant (no DNA synthesis), but may play a regulatory role, as the enzyme shows certain non-catalytic activities, including capacity to bind mRNA (its own and some others) and inhibit translation, and is suspected to be engaged in regulation of several cellular genes [21], as well as to exert an oncogene-like activity [22]. ...
... The latter resin, allowing stronger enzyme binding in the presence of phosphatase inhibitors, was synthesized as earlier described [12], using 5 mg of Raltitrexed (Sigma-Aldrich) per ml of CNBr-activated Sepharose. C. elegans [11] and mouse [13] coding regions were cloned into pPIGDM4 + stop vector and expressed as HisTag-free proteins in BL21(DE3) or a TS-deficient TX61 − (a kind gift from Dr. W. S. Dallas) E. coli strain, respectively. Human [14] and rat [15] TS coding regions were subcloned into pET28a vector and expressed as HisTag-proteins in an E. coli BL21(DE3) strain. ...
... A previously described method was used, with an anti-TS polyclonal antibody [11]. ...
... Interestingly, although protein content and concentration of endogenous TS purified to a specific activity of 1.5 μmol/min/mg protein in the presence of phosphatase inhibitors from C. elegans were too low to perform extensive electrophoretic studies, its catalytic properties differed from those of the endogenous enzyme purified in the absence of phosphatase inhibitors, as previously demonstrated for the L1210 parental and FdUrd-resistant cell enzyme forms [7]. The presence of phosphatase inhibitors in the purification buffers resulted in a product with a K m app for dUMP of 14.5 ± 14% μM (2), hence an order of magnitude higher than that (K m of 1 μM; [11]) for the reaction catalyzed by the C. elegans enzyme purified without the use of inhibitors. ...
... Otóż w przypadku ST inhibicja tego typu demonstrowała dwufazową zależność prędkości inaktywacji od czasu, sugerując różne oddzia-ływanie z dwoma miejscami wiążącymi na cząsteczce enzymu. Można to interpretować jako objaw negatywnej kooperacji [28][29][30][31][32][33][34][35]. ...
... Wyniki naszych badań sugerują jednak zależność od fosforylacji nie tylko aktywności katalitycznej, ale także wspomnianych już wyżej zdolności białka syntazy tymidylanowej do wiązania mRNA i hamowania translacji. Badania te dotyczyły enzymu rekombinowanego, wyprodukowanego w komórkach bakteryjnych i rozdzielonego na formy nieufosforylowaną i ufosforylowaną, przy czym modyfikacja tej ostatniej okazała się dotyczyć tylko reszty/reszt histydynowej/histy- [28,[30][31][32][33][34][35]48,66,[71][72][73][74][75][76]. K i (S-B) -stała hamowania powolnego wiązania (ang. ...
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... Mouse (mTS), C. elegans (CeTS), and T. spiralis (TspTS) thymidylate synthase recombinant proteins were overexpressed and purified as previously described [22,[35][36][37][38], with phosphatase inhibitors (50 mM NaF, 5 mM Na-pyrophosphate, 0.2 mM EGTA, 0.2 mM EDTA and 2 mM Na 3 VO 4 ) present in the purification buffers. TS activity was measured either spectrophotometrically [39] or with the use of the tritium release assay [40]. ...
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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.
... 2.9. Toxicity to Caenorhabditis elegans, a nematode parasite model C. elegans was maintained as previously described (Wińska et al., 2005). The effect of α-mangostin or mebendazole (the latter used as a positive control) on C. elegans population growth was determined according to Simpkin and Coles (1981), with the chlorhexidine prewashing step omitted, by comparing the population levels reached in the control and test wells after 7 days of incubation at 20°C. ...
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... 2.10. Toxicity of 1 to Caenorhabditis elegans, a metazoan model animal C. elegans was maintained as previously described [22]. The effect of a compound on C. elegans population growth was determined according to Simpkin and Coles, with the chlorhexidine prewashing step omitted, by comparing the population levels reached in the control and test wells after 7 day incubation at 20°C [23]. ...
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... Activity remaining after preincubation was determined by addition of 50 mM [5-3 H]dUMP (4 Â 10 4 dpm nmol À1 ) and measurement of tritium release following 4 min incubation. 32 The slopes of the semi-log plots of percent remaining activity vs. preincubation time, expressing apparent inactivation rate constants (k app ) and corresponding inhibitor concentrations ([I]), were then replotted as double-reciprocal plots, and the values of k 2 and K i were determined from the plot intercept and slope, respectively. 44 ...
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... Those studies showed high specific activities of ThyA and other enzymes involved in thymidylate biosynthesis, dUTP-ase and dihydrofolate reductase and ribonucleotide reductase (EC 1.17.4.1 ) to be present ( Table 1 ) in all developmental C. elegans forms (both adult and larval, including developmentally arrested dauer), as had been found for parasitic nematodes T. spiralis . High levels of ThyA mRNA were found throughout nematode development [ 12 ]. ...
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