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ABSTRACT:Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissuespecific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.

ABSTRACT:A cDNA encoding for zebrafish ␥-glutamyl hydrolase (␥GH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-z␥GH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant z␥GH was similar to mammalian ␥GH. Folate is an essential B vitamin and participates in the biosynthesis and metabolism of nucleic acids, proteins, several amino acids, methyl groups, many neurotransmitters, and some vitamins. Mammalian cells are unable to synthesize folates de novo and therefore depend on their food for the supply of folates. Naturally occurring folates are synthesized as poly-␥-glutamate forms (folylpolyglutamate) but are absorbed and transported most efficiently as folylmonoglutamates. The conversion of folylpolyglutamates in dietary food to folylmonoglutamates is catalyzed by carboxypeptidase II (EC 3.4.22.12) in mammals. In a recent study, ␥-glutamyl hydrolase (␥GH, EC 3.4.19.9), a lysosomal cysteine peptidase, was reported to be the enzyme responsible for hydrolyzing dietary folate in rat small intestine (Shafizadeh and Halsted, 2007). After entering cells, these folylmonoglutamates are elongated to folylpolyglutamates by folylpoly-␥-glutamate synthetase (EC 6.3.2.17) for more effective retention and cofactor activity. As part of a salvage pathway, ␥GH catalyzes the hydrolysis of Glu-␥-Glu bonds to form folylmonoglutamates, enabling folate cofactors to be exported from cells and enter circulation again (Suh et al., 2001). Therefore, folylpoly-␥-glutamate synthetase and ␥GH are crucial for the maintenance and regulation of intracellular folate homeostasis. Nevertheless, the mechanism of turnover and control of cellular folylpolyglutamate levels by ␥GH remain unclear. A better understanding of this enzyme and an efficient assay system is prerequisite to answer this question.Consistent with this notion, the activity of ␥GH to hydrolyze the ␥-glutamyl peptide bonds of folylpolyglutamates has rendered this enzyme a potential target of antifolate chemotherapy and, at the same time, a primary component in regulating the intracellular levels of some antifolate drugs. Antifolate drugs, such as methotrexate, owe much of their effectiveness to being substrates for both folylpoly-␥-glutamate synthetase and ␥GH. Removal of ␥-linked glutamate residues decreases the retention and activity of these drugs. A polymorphism resulting in reduced catalytic activity of ␥GH was observed to be associated with greater accumulation of long-chain methotrexate polyglutamate forms (Cheng et al., 2004). In contrast, higher ␥GH activity has been connected to the development of drug resistance (Rhee et al., 1993). Therefore, alteration to ␥GH availability or activity seems to have functional and pharmacological consequences and even to be a potential tumor marker (Schneider and Ryan, 2006). Having pu...

SUMMARYThe etiology of epilepsy is a very complicated, multifactorial process that is not completely understood. Therefore, the availability of epilepsy animal models induced by different mechanisms is crucial in advancing our knowledge and developing new therapeutic regimens for this disorder. Considering the advantages of zebrafish, we have developed a seizure model in zebrafish larvae using ginkgotoxin, a neurotoxin naturally occurring in Ginkgo biloba and hypothesized to inhibit the formation of the neurotransmitter γ-aminobutyric acid (GABA). We found that a 2-hour exposure to ginkgotoxin induced a seizure-like behavior in zebrafish larvae. This seizure-like swimming pattern was alleviated by the addition of either pyridoxal-5′-phosphate (PLP) or GABA and responded quickly to the anti-convulsing activity of gabapentin and phenytoin, two commonly prescribed anti-epileptic drugs (AEDs). Unexpectedly, the ginkgotoxin-induced PLP depletion in our experimental setting did not affect the homeostasis of folate-mediated one-carbon metabolism, another metabolic pathway playing a crucial role in neural function that also relies on the availability of PLP. This ginkgotoxin-induced seizure behavior was also relieved by primidone, which had been tested on a pentylenetetrazole-induced zebrafish seizure model but failed to rescue the seizure phenotype, highlighting the potential use and complementarity of this ginkgotoxin-induced seizure model for AED development. Structural and morphological characterization showed that a 2-hour ginkgotoxin exposure did not cause appreciable changes in larval morphology and tissues development. In conclusion, our data suggests that this ginkgotoxin-induced seizure in zebrafish larvae could serve as an in vivo model for epileptic seizure research and potential AED screening.