Uncommon and parallel developmental patterns of thymidylate synthase expression and localization in Trichinella spiralis and Caenorhabditis elegans

Magdalena Dąbrowska 1 , Barbara Gołos 1 , Elżbieta Wałajtys-Rode 2 , Zbigniew Zieliński 1 , Patrycja Wińska 1 , Joanna Cieśla 1 , Tadeusz Moczoń 3 ,  and Wojciech Rode 1
  • 1 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland
  • 2 Faculty of Chemistry, Warsaw University of Technology, Warszawa, Poland
  • 3 Institute of Parasitology, Polish Academy of Sciences, Warszawa, Poland
Magdalena Dąbrowska, Barbara Gołos, Elżbieta Wałajtys-Rode, Zbigniew Zieliński, Patrycja Wińska, Joanna Cieśla, Tadeusz Moczoń and Wojciech Rode

Abstract

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.

Introduction

Trichinella spiralis is a parasitic nematode causing trichinellosis, a serious disease. Mating of adult worms (developing from infective larvae, deriving from digested infected undercooked meat) occurs in a nonmembrane-bound section of columnar epithelium of the small intestine of the host. Fertilized females enter the intestinal wall and release newborn larvae to the lymph and bloodstream. Each of these penetrates skeletal muscle cell of the host and lives its section, modified in response to the presence of the larva, surrounded by a collagen capsule around which a circulatory rete develops, called the “nurse cell” (Figure 1). Nurse cell development within the muscle cell of the host, initiated by T. spiralis infection, is associated with a variety of changes, including cell cycle re-entry and induction of DNA synthesis, followed by apparent cell cycle arrest, suggested earlier to be of G2/M type [1], but recently identified as a hypermitogenic G1-like arrest [2]. In the nurse cell, the larva will grow and develop, reaching the stage of the infectious form after 15 days [3].

Figure 1
Figure 1

Life cycle of Caenorhabditis elegans (left) and Trichinella spiralis (right). C. elegans larval forms are marked L1–4. C. elegans dauer (German verb “dauern” means “to endure”) larva and T. spiralis muscle larva are developmentally arrested forms. Whereas the former is an optional long-time survival stage of the life cycle, the latter is an infective form, being an obligatory stage of the life cycle.

Citation: Pteridines 24, 1; 10.1515/pterid-2013-0009

Caenorhabditis elegans is a free-living nematode, widely applied as a model in genetic, developmental and biochemical studies, also suggested to be used as such with respect to parasitic nematodes [4]. This small organism, its adult form being self-fertilizing hermaphrodite or male, is characterized by fast development (approx. 3 days at 25°C), involving four larval stages separated by molts, preceding adulthood (Figure 1). Certain conditions, for example, of poorer food supply, will cause formation of dauer larvae, a developmental stage that corresponds to T. spiralis muscle larvae, but, unlike that, is not obligatory [4].

Thymidylate synthase

Thymidylate synthase (EC 2.1.1.45; ThyA) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by N5,10-methylenetetrahydrofolate (meTHF) to generate thymidylate (dTMP) and dihydrofolate. The reaction provides the sole intracellular de novo source of dTMP, making the enzyme essential for regulating the balanced supply of DNA precursors required for DNA replication, and is consequently also a key target for antitumor, antiviral, antifungal and antiprotozoan chemotherapy [5, 6].

High specific activity has been found of ThyA and certain other enzymes engaged in thymidylate biosynthesis, including dihydrofolate reductase (EC 1.5.1.3) and dUTP-ase (EC 3.6.1.23), not only in adult forms but also in muscle larvae of the parasitic nematode, T. spiralis, persisting in the larvae for 2 years after infection [7, 8]. Whereas the presence of ThyA and dUTP-ase in adult worms might be expected (female worms filled with young larvae), expression of the two enzymes in developmentally arrested muscle larvae was surprising. The constant presence of high level of ThyA activity in the larvae, accompanied by enzyme mRNA level similar to that found in newborn larvae and adult worms [9], is an interesting phenomenon, especially as the enzyme protein is suspected of being responsible not only for the catalysis of the key step of thymidylate biosynthesis and regulation of its own translation but also for regulation of other cellular genes [10], and high cellular level of its protein has been ascribed an oncogene-like activity [11]. In particular, a possibility of playing additional, besides catalytic, roles is indicated by a high level of enzyme expression in muscle larvae found later than 38 days after infection (Table 1), that is, at the time of documented lowered thymidine incorporation into larval DNA [15].

Table 1

Specific activities of selected enzymes involved in thymidylate de novo biosynthesis in T. spiralis and T. pseudospiralis muscle larvae, T. spiralis adult forms, C. elegans adult forms, and L1, L3 and dauer larvaea.

Developmental stage (the time between infection and parasite isolation is given)Enzyme specific activity, nmol/min/mg protein
Thymidylate synthaseDihydrofolate reductasedUTPaseRibonucleotide reductase
T. spiralis
 Adult forms
  6 days0.058 6.0NTbNT
 Muscle larvae
  30 days0.092c7.600.250.003
  24 months0.088cNT0.23NT
C. elegans
 Adult forms0.127.43.90.012
 L1 larvae0.1113.22.7NT
 L3 larvae0.407.84.2NT
 Dauer larvae0.10c14.00.830.008

aFor both Trichinella species time between infection and isolation is indicated [8, 12, 13]; bnot tested; ccf. specific activity of 0.1 nmol/min/mg protein found in regenerating rat liver extracts [14].

Moreover, further studies demonstrated a similar phenomenon occurring in the life cycle of the free-living nematode C. elegans. The developmentally arrested C. elegans dauer larvae correspond to the developmentally arrested infective forms, such as Trichinella muscle larvae, of parasitic nematodes [4]. 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].

Thymidylate synthase and cell proliferation

ThyA [16] induction is known to be associated with cell proliferation. A good illustration of the latter is the enzyme specific activity in normal rat liver extracts of 0.003 nmol/min/mg protein [17], thus barely detectable, increasing to 0.1 nmol/min/mg protein [14] in extracts from rat liver regenerating following partial hepatectomy. Of note is that liver regeneration is a model of fast proliferating tissue.

Careful comparison of ThyA levels in proliferating and confluent cells showed the absence of detectable enzyme in the latter [18], indicating its induction in cells to be due to leaving quiescence (cell cycle G0 phase). Besides, in mammalian cells ThyA mRNA level is very low in the G0 phase, increases by 10- to 20-fold when cells enter S phase [19, 20], again becoming lower in the course of differentiation [21]. Comparison of different cells and tissues showed ThyA mRNA level to vary, reflecting the differences in proliferation rate [22]. Therefore, the presence of high level of the enzyme and its mRNA throughout the development of each nematode, and especially its persistence in developmentally arrested forms, that is, T. spiralis muscle larva and C. elegans dauer larva, is particularly unexpected, as it suggests the presence of large populations of cycling cells. With respect to the latter, it should be noted that extracts from those developmentally arrested forms show ThyA specific activity similar to that found in regenerating rat liver extracts (Table 1). To explain this, at least in the case of the parasite, the muscle larva cell population may be assumed to be undergoing cell cycle arrest through the lifetime of the host [7, 8].

Global cell cycle arrest in nematodes

In accordance with foregoing ThyA immunolocalization during development of T. spiralis and C. elegans with the use of confocal microscopy revealed the presence of high enzyme levels in developing embryos, gonad primordia, egg and sperm cells, nerve ring and secretory cells, opening to T. spiralis esophagus (Figure 2) and C. elegans pharynx [23]. With the embryos, such distribution of the enzyme, known to be associated with proliferating tissues, is not unexpected. As high levels of ThyA are also known to characterize certain cell cycle-arrested biological systems, for example, unfertilized eggs [24–26], with animal oocytes shown to undergo cell cycle arrest before fertilization [27], its presence in the egg and sperm cells, nerve ring, as well as in secretory cells of both species points to those cell populations remaining cell cycle arrested.

Figure 2
Figure 2

Thymidylate synthase immunolocalization (reflected by fluorescence signal) during Caenorhabditis elegans (1, 3 and 7) and Trichinella spiralis (2, 4–6, 8–10) development [21]. (1) Distinct fluorescence signal in the nerve ring (arrowhead) of a 465 μm-long C. elegans L3 larva. (2) An 800 μm-long premature T. spiralis muscle larva: magnification of the nerve ring showing strong fluorescence. (3) Fluorescence signal in the gonad primordia (along the body wall) of the larva shown in (1). (4) Clear fluorescence signal in the uterus primordium (arrowhead) and ovary primordium (arrow) of a female T. spiralis muscle larva. (5) High fluorescence signal in or around the nuclei of the excretory-secretory organ stichosome (arrowhead) and in gonad primordium (arrow) of a male T. spiralis muscle larva. (6) Stichosome of an adult female T. spiralis, with high fluorescence signal in the nuclear region (the arrows indicate positions of nuclei laying out of focus). (7) An enlarged image of strong fluorescence signal in embryos developing in an adult C. elegans hermaphrodite. (8) A magnified image of embryos developing in the uterus of an adult T. spiralis female and showing a distinct signal. (9) An in silico magnified image of the seminal vesicle with a clear fluorescence signal from secondary spermatocytes of a male T. spiralis adult. (10) Strong signals demonstrated by T. spiralis larvae before birth.

Citation: Pteridines 24, 1; 10.1515/pterid-2013-0009

The results suggest unusual regulation of the cell cycle in nematodes, manifested by a global cell cycle arrest in developmentally arrested larvae, such as T. spiralis muscle larvae and C. elegans dauer; the latter interpretation also being supported by Hong et al. [28] who drew a similar conclusion about global cell cycle arrest in dauer larvae based on different symptoms.

It should be mentioned that the above presented phenomenon of global cell cycle arrest in the developmentally arrested larvae, such as T. spiralis muscle larvae and C. elegans dauer larvae, may be potentially exploitable as a target for selective chemotherapy aimed against parasitic nematodes. Especially as ThyA protein may play some regulatory roles. Learning a possibility of selective influence on this protein in nematode cells (e.g., via change of conformation resulting from inhibitor binding) should enable interference with those functions.

Supported by the National Science Center Grant No. 2011/01/B/NZ6/01781.

References

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    Jasmer DP. Trichinella spiralis infected muscle cells arrest in G2/M and cease muscle gene expression. J Cell Biol 1993;121:785–93.

    • Crossref
  • 2.

    Dąbrowska M, Skoneczny M, Zieliński Z, Rode W. Nurse cell of Trichinella spp. as a model of long-term cell cycle arrest. Cell Cycle 2008;7:2167–78.

    • Crossref
    • PubMed
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    Despommier DD. How does Trichinella spiralis make itself at home? Parasitol Today 1998;14:318–23.

    • Crossref
    • PubMed
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    Bürglin TR, Labos E, Blaxter ML. Caenorhabditis elegans as a model for parasitic nematodes. Int J Parasitol 1998;28:395–411.

    • Crossref
    • PubMed
  • 5.

    Rode W, Leś A. Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and/or 4-substituted analogues of dUMP and 5-fluoro-dUMP. Acta Biochim Pol 1996;43:133–42.

    • Crossref
    • PubMed
  • 6.

    Costi MP, Ferrari S, Venturelli A, Calò S, Tondi D, Barlocco D. Thymidylate synthase structure, function and implication in drug discovery. Curr Med Chem 2005;12:2241–58.

    • Crossref
    • PubMed
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    Dąbrowska M, Zieliński Z, Wranicz M, Michalski R, Pawełczak K, Rode W. Trichinella spiralis thymidylate synthase: developmental pattern, isolation, molecular properties, and inhibition by substrate and cofactor analogues. Biochem Biophys Res Commun 1996;228:440–5.

    • Crossref
  • 8.

    Rode W, Dąbrowska M, Zielinski Z, Gołos B, Wranicz M, Felczak K, et al. Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage. Parasitology 2000;120:593–600.

    • Crossref
  • 9.

    Dąbrowska M, Jagielska E, Cieśla J, Płucienniczak A, Kwiatowski J, Wranicz M, et al. Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression. Parasitology 2004;128:209–21.

    • Crossref
  • 10.

    Liu J, Schmitz JC, Lin X, Tai N, Yan W, Farrell M, et al. Thymidylate synthase as a translational regulator of cellular gene expression. Biochim Biophys Acta 2002;1587:174–82.

    • Crossref
    • PubMed
  • 11.

    Rahman L, Voeller D, Rahman M, Lipkowitz S, Allegra C, Barrett JC, et al. Thymidylate synthase as an oncogene: a novel role for an essential DNA synthesis enzyme. Cancer Cell 2004;5:341–51.

    • Crossref
    • PubMed
  • 12.

    Wińska P, Gołos B, Cieśla J, Zieliński Z, Frączyk T, Wałajtys-Rode E, et al. Developmental arrest in C. elegans dauer larvae leaves high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae. Parasitology 2005;131:247–54.

    • Crossref
  • 13.

    Dąbrowska M, Gołos B, Wałajtys-Rode E, Wińska P, Cieśla J, Zieliński Z, et al. Unusual developmental pattern of expression of enzymes involved in DNA biosynthesis in Trichinella spiralis and Trichinella pseudospiralis. In: Viola-Magni M, editor. Detection of bacteria, viruses, parasites and fungi. Dordrecht: Springer, 2010:333–56.

  • 14.

    Cieśla J, Gołos B, Dzik JM, Pawełczak K, Kempny M, Makowski M, et al. Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues. Biochim Biophys Acta 1995;1249:127–36.

    • Crossref
    • PubMed
  • 15.

    Stewart GL, Read CP. Deoxyribonucleic acid metabolism in mouse trichinosis. J Parasitol 1973;59:264–7.

    • Crossref
  • 16.

    Santi DV, Danenberg PV. Folates in pyrimidine biosynthesis. In: Blakley RL, Benkovic SJ, editors. Folates and pterines, vol. 1. New York: Wiley, 1984:345–98.

  • 17.

    Labow R, Maley GF, Maley F. The effect of methotrexate on enzymes induced following partial hepatectomy. Cancer Res 1969;29:366–72.

    • PubMed
  • 18.

    Pestalozzi BC, McGinn CJ, Kinsella TJ, Drake JC, Glennon MC, Allegra CJ, et al. Increased thymidylate synthase protein levels are principally associated with proliferation but not cell cycle phase in asynchronous human cancer cells. Br J Cancer 1995;71:1151–7.

    • Crossref
  • 19.

    Ayusawa D, Shimizu K, Koyama H, Kaneda S, Takeishi K, Seno T. Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol 1986;190:559–67.

    • Crossref
  • 20.

    Gribaudo G, Riera L, Rudge TL, Caposio P, Johnson LF, Landolfo S. Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts. J Gen Virol 2002;83:2983–93.

    • Crossref
  • 21.

    Horie N, Nozawa R, Takeishi K. Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase. Biochem Biophys Res Commun 1992;185:127–33.

    • Crossref
  • 22.

    Lee Y, Shen G, Johnson LF. Complex transcriptional initiation pattern of the thymidylate synthase promoter in mouse tissues. Archiv Biochem Biophys 1999;372:389–92.

    • Crossref
  • 23.

    Gołos B, Dąbrowska M, Wałajtys-Rode E, Zieliński Z, Wińska P, Cieśla J, et al. Immunofluorescent localization of thymidylate synthase in the development of Trichinella spiralis and Caenorhabditis elegans. Mol Biochem Parasitol 2012;183: 63–9.

    • Crossref
  • 24.

    Carpenter NJ. Thymidylate synthetase in mutants of Drosophila melanogaster. Genetics 1973;75:113–22.

  • 25.

    Rode W, Szymanowska H. Developmental pattern of thymidylate synthetase activity in silkworm: Bombyx mori L. Insect Biochem 1976;6:333–7.

    • Crossref
  • 26.

    Yasumasu I, Saitoh M, Fujimoto N, Kusunoki S. Changes in activities of thymidylate synthetase and dihydrofolate reductase in sea urchin eggs after fertilization. Dev Growth Differ 1979;21:237–43.

    • Crossref
  • 27.

    Sagata N. Meiotic mataphase arrest in animal oocytes: its mechanisms and biological significance. Trends Cell Biol 1996;6:22–8.

  • 28.

    Hong Y, Roy R, Ambros V. Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans. Development 1998;125:3585–97.

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  • 1.

    Jasmer DP. Trichinella spiralis infected muscle cells arrest in G2/M and cease muscle gene expression. J Cell Biol 1993;121:785–93.

    • Crossref
  • 2.

    Dąbrowska M, Skoneczny M, Zieliński Z, Rode W. Nurse cell of Trichinella spp. as a model of long-term cell cycle arrest. Cell Cycle 2008;7:2167–78.

    • Crossref
    • PubMed
  • 3.

    Despommier DD. How does Trichinella spiralis make itself at home? Parasitol Today 1998;14:318–23.

    • Crossref
    • PubMed
  • 4.

    Bürglin TR, Labos E, Blaxter ML. Caenorhabditis elegans as a model for parasitic nematodes. Int J Parasitol 1998;28:395–411.

    • Crossref
    • PubMed
  • 5.

    Rode W, Leś A. Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and/or 4-substituted analogues of dUMP and 5-fluoro-dUMP. Acta Biochim Pol 1996;43:133–42.

    • Crossref
    • PubMed
  • 6.

    Costi MP, Ferrari S, Venturelli A, Calò S, Tondi D, Barlocco D. Thymidylate synthase structure, function and implication in drug discovery. Curr Med Chem 2005;12:2241–58.

    • Crossref
    • PubMed
  • 7.

    Dąbrowska M, Zieliński Z, Wranicz M, Michalski R, Pawełczak K, Rode W. Trichinella spiralis thymidylate synthase: developmental pattern, isolation, molecular properties, and inhibition by substrate and cofactor analogues. Biochem Biophys Res Commun 1996;228:440–5.

    • Crossref
  • 8.

    Rode W, Dąbrowska M, Zielinski Z, Gołos B, Wranicz M, Felczak K, et al. Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage. Parasitology 2000;120:593–600.

    • Crossref
  • 9.

    Dąbrowska M, Jagielska E, Cieśla J, Płucienniczak A, Kwiatowski J, Wranicz M, et al. Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression. Parasitology 2004;128:209–21.

    • Crossref
  • 10.

    Liu J, Schmitz JC, Lin X, Tai N, Yan W, Farrell M, et al. Thymidylate synthase as a translational regulator of cellular gene expression. Biochim Biophys Acta 2002;1587:174–82.

    • Crossref
    • PubMed
  • 11.

    Rahman L, Voeller D, Rahman M, Lipkowitz S, Allegra C, Barrett JC, et al. Thymidylate synthase as an oncogene: a novel role for an essential DNA synthesis enzyme. Cancer Cell 2004;5:341–51.

    • Crossref
    • PubMed
  • 12.

    Wińska P, Gołos B, Cieśla J, Zieliński Z, Frączyk T, Wałajtys-Rode E, et al. Developmental arrest in C. elegans dauer larvae leaves high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae. Parasitology 2005;131:247–54.

    • Crossref
  • 13.

    Dąbrowska M, Gołos B, Wałajtys-Rode E, Wińska P, Cieśla J, Zieliński Z, et al. Unusual developmental pattern of expression of enzymes involved in DNA biosynthesis in Trichinella spiralis and Trichinella pseudospiralis. In: Viola-Magni M, editor. Detection of bacteria, viruses, parasites and fungi. Dordrecht: Springer, 2010:333–56.

  • 14.

    Cieśla J, Gołos B, Dzik JM, Pawełczak K, Kempny M, Makowski M, et al. Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues. Biochim Biophys Acta 1995;1249:127–36.

    • Crossref
    • PubMed
  • 15.

    Stewart GL, Read CP. Deoxyribonucleic acid metabolism in mouse trichinosis. J Parasitol 1973;59:264–7.

    • Crossref
  • 16.

    Santi DV, Danenberg PV. Folates in pyrimidine biosynthesis. In: Blakley RL, Benkovic SJ, editors. Folates and pterines, vol. 1. New York: Wiley, 1984:345–98.

  • 17.

    Labow R, Maley GF, Maley F. The effect of methotrexate on enzymes induced following partial hepatectomy. Cancer Res 1969;29:366–72.

    • PubMed
  • 18.

    Pestalozzi BC, McGinn CJ, Kinsella TJ, Drake JC, Glennon MC, Allegra CJ, et al. Increased thymidylate synthase protein levels are principally associated with proliferation but not cell cycle phase in asynchronous human cancer cells. Br J Cancer 1995;71:1151–7.

    • Crossref
  • 19.

    Ayusawa D, Shimizu K, Koyama H, Kaneda S, Takeishi K, Seno T. Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol 1986;190:559–67.

    • Crossref
  • 20.

    Gribaudo G, Riera L, Rudge TL, Caposio P, Johnson LF, Landolfo S. Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts. J Gen Virol 2002;83:2983–93.

    • Crossref
  • 21.

    Horie N, Nozawa R, Takeishi K. Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase. Biochem Biophys Res Commun 1992;185:127–33.

    • Crossref
  • 22.

    Lee Y, Shen G, Johnson LF. Complex transcriptional initiation pattern of the thymidylate synthase promoter in mouse tissues. Archiv Biochem Biophys 1999;372:389–92.

    • Crossref
  • 23.

    Gołos B, Dąbrowska M, Wałajtys-Rode E, Zieliński Z, Wińska P, Cieśla J, et al. Immunofluorescent localization of thymidylate synthase in the development of Trichinella spiralis and Caenorhabditis elegans. Mol Biochem Parasitol 2012;183: 63–9.

    • Crossref
  • 24.

    Carpenter NJ. Thymidylate synthetase in mutants of Drosophila melanogaster. Genetics 1973;75:113–22.

  • 25.

    Rode W, Szymanowska H. Developmental pattern of thymidylate synthetase activity in silkworm: Bombyx mori L. Insect Biochem 1976;6:333–7.

    • Crossref
  • 26.

    Yasumasu I, Saitoh M, Fujimoto N, Kusunoki S. Changes in activities of thymidylate synthetase and dihydrofolate reductase in sea urchin eggs after fertilization. Dev Growth Differ 1979;21:237–43.

    • Crossref
  • 27.

    Sagata N. Meiotic mataphase arrest in animal oocytes: its mechanisms and biological significance. Trends Cell Biol 1996;6:22–8.

  • 28.

    Hong Y, Roy R, Ambros V. Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans. Development 1998;125:3585–97.

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  • View in gallery

    Life cycle of Caenorhabditis elegans (left) and Trichinella spiralis (right). C. elegans larval forms are marked L1–4. C. elegans dauer (German verb “dauern” means “to endure”) larva and T. spiralis muscle larva are developmentally arrested forms. Whereas the former is an optional long-time survival stage of the life cycle, the latter is an infective form, being an obligatory stage of the life cycle.

  • View in gallery

    Thymidylate synthase immunolocalization (reflected by fluorescence signal) during Caenorhabditis elegans (1, 3 and 7) and Trichinella spiralis (2, 4–6, 8–10) development [21]. (1) Distinct fluorescence signal in the nerve ring (arrowhead) of a 465 μm-long C. elegans L3 larva. (2) An 800 μm-long premature T. spiralis muscle larva: magnification of the nerve ring showing strong fluorescence. (3) Fluorescence signal in the gonad primordia (along the body wall) of the larva shown in (1). (4) Clear fluorescence signal in the uterus primordium (arrowhead) and ovary primordium (arrow) of a female T. spiralis muscle larva. (5) High fluorescence signal in or around the nuclei of the excretory-secretory organ stichosome (arrowhead) and in gonad primordium (arrow) of a male T. spiralis muscle larva. (6) Stichosome of an adult female T. spiralis, with high fluorescence signal in the nuclear region (the arrows indicate positions of nuclei laying out of focus). (7) An enlarged image of strong fluorescence signal in embryos developing in an adult C. elegans hermaphrodite. (8) A magnified image of embryos developing in the uterus of an adult T. spiralis female and showing a distinct signal. (9) An in silico magnified image of the seminal vesicle with a clear fluorescence signal from secondary spermatocytes of a male T. spiralis adult. (10) Strong signals demonstrated by T. spiralis larvae before birth.