Oxidative stress is one of the major characteristics in Alzheimer’s disease, and converging evidence indicates that cysteine S-nitrosylation might be related in AD pathology. My results demonstrated exogenous S-nitrosoglutathione was able to S-nitrosylate vAChT, vMAT2, vGluT1 and vGluT2. S-nitrosylation of these vesicular transporters inhibited the uptake of [3H]acetylcholine, [3H]dopamine and [3H]glutamate respectively. APP/PS1 transgenic mice were used to investigate neurotransmission dysfunctions of Alzheimer’s disease. Global protein S-nitrosylation was increased in the 9 and 12 month APP/PS1 mice. Further investigation demonstrated an increase of vAChT and vGluT1 S-nitrosylation in frontal cortex of 6, 9 and 12 month APP/PS1 mice and an increased vAChT and vGluT1 S-nitrosylation was found in hippocampus of 3 month APP/PS1 mice. These findings together suggest that S-nitrosylation of vesicular transporters inhibits the uptake of neurotransmitters, and S-nitrosylation of vAChT and might be associated with the neurotransmission dysfunction of acetylcholine and glutamate in Alzheimer’s disease.

The enrichment of egg with folate is a viable option for supplying the general population of a food product rich in natural folates. However, attempts to increase the concentration of folate in egg beyond the achieved level of enrichment had been unsuccessful because egg folate reached a maximum plateau when folic acid (FA) was increased in the diet. Thus, experiments were conducted to determine the factors regulating the deposition of dietary FA into the eggs. In the first study, the effect of feeding equimolar intake of FA and 5-methyltetrahydrofolate (5-methylTHF), the biologically active form of folate; on egg folate concentrations, indices of folate status, and activities of folate-dependent enzymes was evaluated. Folic acid and 5-methylTHF demonstrated equivalent effects in enhancing the egg folate concentrations and improving the indices of folate status in the laying hen. The activities of folate-dependent enzymes were similar between the two forms of folate except for hepatic dihydrofolate reductase (DHFR) activity which increased in FA-fed birds compared to 5-methylTHF-fed birds. However, this demonstrated the ability of the laying hen to metabolically convert FA into its biologically active forms. Therefore, the influence of intestinal FA absorption in the regulation of FA deposition in the egg was subsequently evaluated. Using the in vitro everted intestinal sac technique, FA was absorbed in all regions of the intestine. Absorption was maximum at acidic pH 6.0, and increased in the duodenum and jejunum compared to the ileum and cecum. The rate of FA absorption in the jejunum diminished at higher FA concentrations. Therefore, further study was conducted to determine the regulation of FA absorption when levels of FA in the laying hen diet are increased. Supplementation of increased FA levels resulted to a down-regulation of FA absorption in the duodenum, but not in the jejunum of the laying hen. This down-regulation was not associated to a decreased mRNA gene expression of the duodenal folate transporters. Overall, decreased intestinal rate of FA absorption possibly associated to a post-transcriptional or translational regulation of specific folate transporters in the intestine of the laying hen may contribute to the saturation in the egg folate concentration.

Multidrug resistance refers to the simultaneous resistance to structurally and mechanistically unrelated cytotoxic drugs. Chronic administration of cytotoxic drugs to patients with metastatic breast cancer results in the development of multidrug resistance, thus rendering chemotherapy unsuccessful. One mechanism by which multidrug resistance is conferred is the decreased intracellular drug accumulation due to the upregulation of the ATP-binding cassette (ABC) transporters. Jadomycins are polyketide-derived natural products produced by the soil actinomycetes Streptomyces venezuelae, ISP 5230. Jadomycins exhibit anticancer, antibacterial and antifungal activities. Pilot work in our laboratory demonstrated that jadomycin B exhibited similar cytotoxic effects in drug-sensitive and drug-resistant cancer cells. We hypothesize that jadomycins are poor substrates of ABCB1, ABCC1 and ABCG2 efflux transporters, and consequently will exhibit higher intracellular accumulation, which results in improved cytotoxic efficacy over existing chemotherapeutics that are rapidly effluxed by ABC transporters. Using methyltetrazolium (MTT) cell viability assays, the cytotoxic efficacy of nine jadomycin analogues (DNV, L, B, SPhG, F, W, S, T and N) in drug-sensitive and drug-resistant MCF7 breast cancer cells was evaluated. Jadomycin B, L, S and T were found to be equally toxic to drug-sensitive and drug-resistant ABCB1, ABCC1 or ABCG2-overexpressing MCF7 breast cancer cells. The inhibition of ABCB1, ABCC1 or ABCG2 efflux transporters with verapamil, MK-571 or ko143, respectively, did not significantly augment the cytotoxic effects of jadomycin DNV, L, B and S in drug-resistant MCF7 cells, suggesting that these jadomycins are poor substrates of the targeted transporter. Furthermore, all nine jadomycin analogues did not increase the intracellular accumulation of ABCB1, ABCC1 or ABCG2 probe fluorescent substrates in HEK-293 cells, indicating that these jadomycins do not inhibit the efflux function of the transporters. We conclude that jadomycins B, L and S are effective agents in the eradication of resistant breast cancer cells grown in culture, and that the ability of specific jadomycins to retain cytotoxic efficacy in resistant cells stems from their limited interactions with ABCB1, ABCC1 or ABCG2 efflux transporters.

[Truncated abstract] Some plants, such as legumes, are able to use atmospheric nitrogen as a nitrogen source due to the nitrogen-fixing bacteria residing in specialised root structures called nodules. The exchange of carbon and nitrogen between the host plant (legume) and the nitrogen- fixing micro-symbiont is vital for biological nitrogen fixation. In particular, transport of C4-dicarboxylates, mainly malate, from the plant to the micro-symbiont, and the reverse transport of fixed nitrogen in the form of ammonium are essential for symbiotic nitrogen fixation. In the legume nodule, the symbiosome membrane (SM) surrounds the bacteroid and all exchanges of metabolites and nutrients that occur between the plant and the micro-symbiont must cross this membrane. Recently it has been established that cycling of amino acids across the SM is also critical for optimal symbiotic nitrogen fixation. Therefore to fully understand this agriculturally significant phenomenon, the mechanisms facilitating these exchanges need to be investigated. The major aim of this study was to increase the understanding of nutrient exchange within the nodule at the molecular level by isolating and characterising genes encoding transporters responsible for malate and amino acids transport in soybean (Glycine max, L.), with particular interest in genes significantly or highly expressed in nodules. A combination of molecular and biochemical techniques was used to achieve this. ... Southern blot analysis showed that a small gene family of up to five members encodes these proteins in soybean. A full-length cDNA, designated GmAAP5, was isolated that encodes a novel, putative amino acid transporter. Molecular characterisation of this cDNA and that of GmAAP1 (GenBank Accession no: AY029352), a previously identified putative amino acid transporter gene, was done. Expression analyses showed relatively high expression of GmAAP5 in soybean nodules compared to that in leaf and root tissues, while GmAAP1 showed uniformly high expression in root, leaf and nodule tissues. Phylogenetic analysis of the deduced amino acid sequences of known functional AAPs from dicotyledonous plants revealed that GmAAP1 is most closely related to AAP2 from V. faba, while GmAAP5 is more closely related to AAPs from non-leguminous plants than from leguminous plants. Based on the functional characterisation of the AAPs with which GmAAP1 and GmAAP5 cluster, it is likely that both transporters are neutral and acidic amino acid transporters within the AAP subfamily.

Diss. Stockholm : Stockholms universitet, 2005. / S. 312-323: Bibliografi.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 164-175).

Regensburg, Univ., Diss., 2009.

Regensburg, Univ., Diss., 2004.

University, Diss., 2003--Frankfurt (Main).

Techn. Universiẗat, Diss., 2004--Darmstadt.