Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations. The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. Another example of stable resistance can also be seen in sulfonamide resistant Streptococcus pyogenes. Sequence determinations of the dihydropteroate synthase (dhps) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213. The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (hppk). In the malaria parasite Plasmodium falciparum this enzyme is part of a polyfunctional entity, also encoding dhps. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to P. falciparum. Both are crucial for enzyme activity. To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

The hypothesis that high multivitamin gestational (HV) diets affect the development of central serotonergic regulatory systems in female offspring, and that this is due to its high folic acid content, was investigated. Dams were fed the AIN-93G diet containing the recommended multivitamin mix (RV), 10-fold the RV amount (HV), or the RV diet with 10-fold the folic acid (Hfol). Serotonergic control of food intake and macronutrient selection was assessed by measuring long-term intake and selection, short-term intake and selection following injections of serotonin receptor agonists, and hypothalamic serotonin receptor expression. Offspring from Hfol dams selected more protein and were less responsive to agonist injections, but showed no altered serotonin receptor expression. In contrast, those born to HV fed dams were not different from the RV controls in any measure. In conclusion, the Hfol, but not HV gestational diet affects serotonergic regulation of food intake in female rat offspring.

The hypothesis that high multivitamin gestational (HV) diets affect the development of central serotonergic regulatory systems in female offspring, and that this is due to its high folic acid content, was investigated. Dams were fed the AIN-93G diet containing the recommended multivitamin mix (RV), 10-fold the RV amount (HV), or the RV diet with 10-fold the folic acid (Hfol). Serotonergic control of food intake and macronutrient selection was assessed by measuring long-term intake and selection, short-term intake and selection following injections of serotonin receptor agonists, and hypothalamic serotonin receptor expression. Offspring from Hfol dams selected more protein and were less responsive to agonist injections, but showed no altered serotonin receptor expression. In contrast, those born to HV fed dams were not different from the RV controls in any measure. In conclusion, the Hfol, but not HV gestational diet affects serotonergic regulation of food intake in female rat offspring.

Statement of problem: We have shown that the C677T 5,10 methylenetetrahydrofolate reductase (MTHFR) variant is associated with elevated maternal serum α-fetoprotein (MSAFP), the most common screening test for neural tube defects (NTD). Therefore, past contradictory studies of NTDs and C677T MTHFR may have been biased because of changes in case populations after prenatal diagnosis and termination of pregnancy. Further, an unexplained elevation of MSAFP is known to increase the risk for later pregnancy complications. Is the C677T MTHFR variant a predisposing genetic variant for both NTDs and later complications of pregnancy? Methods: A retrospective study of women with pregnancies resulting in NTD outcome and women with unexplained elevations of MSAFP was undertaken. Women and their partners were genotyped for the C677T MTHFR allele. Couples with a pregnancy resulting in a NTD outcome were compared to couples whose pregnancy outcome did not involve. Couples with unexplained elevations of MSAFP who did and did not have later complications of pregnancy were also compared. Allele frequencies for all groups were then compared against the previously established Manitoba population allele frequency (based on 977 consecutive newborn metabolic screening bloodspots). A review of all studies of NTDs and association with the C677T MTHFR variant was undertaken to determine if the association between the variant and MSAFP is a source of bias. NTD incidence was examined before and after folic acid food fortification introduced in Canada in 1999. Results: There is an increase in the allele frequency of the C677T MTHFR variant in parents with an unexplained elevated MSAFP followed by later complications of pregnancy. The C677T MTHFR variant is also a contributing genetic factor to NTDs worldwide. The incidence of NTDs in Manitoba has decreased by 37% since food fortification with folic acid was introduced. Conclusions: The C677T MTHFR variant is a contributing genetic factor to both later complications of pregnancy after an unexplained elevation of MSAFP and to NTDs. This variant is folate sensitive and folic acid fortification has reduced the incidence of NTDs. / February 2005

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine. / text

Porphyrins represent one of the oldest, most widely studied chemical structures, both in nature and in biomedical applications. Due to their tumor avidity and favorable photophysical properties, such as long wavelength absorption and emission, easy derivatization, high singlet oxygen quantum yield and low in vivo toxicity, porphyrins have found particular success for photodynamic therapy and fluorescence imaging of cancer. Additionally, they are excellent metal chelators, forming highly stable metallo-complexes, making porphyrins an efficient delivery vehicle for radioisotopes. Thus, there is great potential in the applications of these multi-modal porphyrin-based agents for cancer imaging and therapy. I have investigated the characteristics of various porphyrin-based probes and their potential application in different clinically relevant models. Here, I will discuss three types of porphyrin-based agents: 1) photodynamic molecular beacons (PPMMPB), 2) targeted peptide porphyrins (PPF) and 3) porphyrin-lipid nanovesicles, porphysomes. I will demonstrate that all of these porphyrin-based agents have potential clinical applications in various fields of cancer imaging and therapy. Although these three agents differ greatly, they all aim to increase the signal-to-background ratio of tumor to healthy tissue uptake of porphyrins, thereby increasing our ability to detect tumor tissue and better preserve healthy tissue during therapy.

Porphyrins represent one of the oldest, most widely studied chemical structures, both in nature and in biomedical applications. Due to their tumor avidity and favorable photophysical properties, such as long wavelength absorption and emission, easy derivatization, high singlet oxygen quantum yield and low in vivo toxicity, porphyrins have found particular success for photodynamic therapy and fluorescence imaging of cancer. Additionally, they are excellent metal chelators, forming highly stable metallo-complexes, making porphyrins an efficient delivery vehicle for radioisotopes. Thus, there is great potential in the applications of these multi-modal porphyrin-based agents for cancer imaging and therapy. I have investigated the characteristics of various porphyrin-based probes and their potential application in different clinically relevant models. Here, I will discuss three types of porphyrin-based agents: 1) photodynamic molecular beacons (PPMMPB), 2) targeted peptide porphyrins (PPF) and 3) porphyrin-lipid nanovesicles, porphysomes. I will demonstrate that all of these porphyrin-based agents have potential clinical applications in various fields of cancer imaging and therapy. Although these three agents differ greatly, they all aim to increase the signal-to-background ratio of tumor to healthy tissue uptake of porphyrins, thereby increasing our ability to detect tumor tissue and better preserve healthy tissue during therapy.

Folic acid is a B vitamin involved in DNA CpG methylation. Mandated dietary fortification has led to a subsequent increase in blood folate concentration which has been correlated to a simultaneous spike in colorectal cancer incidence in Canada and the US. Several human colorectal cancer cell lines were cultivated under low (0 mg/L), standard (4 mg/L), and high (16 mg/L) folate conditions for seven days, then assessed for DNA methyltransferase1 protein expression, changes in DNA methylation, and ability to generate colonospheres in culture. Low folic acid levels generally led to reduced DNMT1 protein expression, CpG hypomethylation, and reduced colonosphere yield. High folic acid levels led to increased DNMT1 protein expression, CpG hypermethylation, and maintained colonosphere yield. This data demonstrates that varying levels of folic acid in vitro can influence the methylation status and cancer stem cell self-renewal ability of human colorectal cancer cells. / Canadian Cancer Society