Folate, vitamin B12, vitamin B6 and riboflavin interact by functioning as cofactors within one-carbon metabolism (OCM), a network of interrelated cellular pathways essential for numerous biological processes, including the biosynthesis of DNA, amino acid interconversions and methylation reactions. The pathways of OCM are influenced by endocrine signals and genetic polymorphisms and are particularly responsive to relevant B-vitamin intakes. Physiological changes in healthy pregnancy, leading to a steady decline in B-vitamin status, add another layer of complexity to the regulation of OCM. Although significant advances have been made to improve our understanding of these pregnancy-related changes, no specific reference ranges yet exist for B-vitamin biomarkers in pregnancy to support normal fetal growth without depleting maternal stores. The lack of pregnancy-related criteria for adequacy of B-vitamin status is in turn a major limitation in identifying pregnant women most at risk of B-vitamin deficiency. Another challenge is that the evidence is very limited to provide a basis for establishing pregnancy-specific dietary recommendations for B-vitamins to support successful pregnancy outcomes. In terms of preventing adverse outcomes, periconceptional folic acid supplementation has a proven role, established more than 30 years ago, in protecting against neural tube defect-affected pregnancies and this has been the major focus of public health policy worldwide. This review evaluates the emerging evidence for the less well recognised role of B-vitamins in preventing hypertensive disorders in pregnancy and the intergenerational effects of B-vitamins on offspring neurodevelopment and cognitive performance during childhood. We also consider the underlying biological mechanisms.

Iron (Fe) status among healthy male and female blood donors, aged 18–65 years, is estimated. General characteristics and lifestyle factors, dietary habits and major one-carbon metabolism-related polymorphisms were also investigated. An explorative cross-sectional study design was used to examine a sample of blood donors attending the Transfusion Medicine Unit of the Verona University Hospital, Italy. From April 2016 to May 2018, 499 subjects were enrolled (255 men, 244 women, 155 of whom of childbearing age). Major clinical characteristics including lifestyle, dietary habits and Fe status were analysed. The MTHFR 677C > T, cSHMT 1420C > T, DHFR 19bp ins/del and RFC1 80G > A polymorphisms were also assayed. Mean plasma concentrations of Fe and ferritin were 16·6 µmol/l (95 % CI 16·0, 17·2) and 33·8 µg/l (95 % CI 31·5, 36·2), respectively. Adequate plasma Fe concentrations (> 10·74 µmol/l) were detected in 84·3 % and adequate ferritin concentrations (20–200 µg/l) was found in 72·5 % of the whole cohort. Among the folate-related polymorphisms analysed, carriers of the DHFR 19bp del/del mutant allele showed lower ferritin concentration when compared with DHFR 19bp ins/del genotypes. In a sample of Italian healthy blood donors, adequate plasma concentrations of Fe and ferritin were reached in a large proportion of subjects. The relationship of Fe status with lifestyle factors and folate-related polymorphisms requires more investigation to clarify further gene–nutrient interactions between folate and Fe metabolism.

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.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent antimalarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 (‘Human’) from 2 (‘Cattle’) in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. falciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (AGC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2.5×10−9 mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and anti-malarial drug therapy by demonstrating that even with low mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from 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. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.

Recent developments on virus-like particles have demonstrated their potential in transfecting eucaryotic cells. In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids. Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting. Generation of a cell-type specificity of this system would imply the presentation of a new function on the surface of VP1. To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein. The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively. The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion. The inserted dihydrofolate reductase showed major changes when compared to the wild-type form. The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased KM values for its substrates. This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties.

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N-labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein-ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 °C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower-temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4-diaminopyrimidine-containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side-chain orientations and local conformations of which are not greatly changed in the different complexes (similar χ1 and χ2 values).

The effects of folate sufficiency and deficiency in three pathways of folate metabolism were studied in 2- and 3-week-old broiler chicks. Erythrocyte phosphoribosylpyrophosphate concentrations and dihydro- folate reductase (EC 1.5.1.3) activity were significantly elevated in folate deficiency. Percentage incorporation of deoxyuridine into bone marrow DNA was reduced in folate deficiency. There was a trend towards reduced liver dihydrofolate reductase activity in deficient chicks. These studies identify further biochemical criteria that can be used to assess folate status of chicks.