Choline is known to be essential for brain development and neural function, but its impact on the retina, as a type of neural tissue, is unknown. This study examined the effects of choline during fetal development on membrane phospholipid (PL) compositions and functions in neural tissues, brain and retina. Pregnant C57 BL/6 mice were fed one of the 4 choline modified diets from gestation to early development: i) deficient (Def, 0g/kg), ii) control (Cont, 2.5g/kg), iii) supplemented with choline chloride (Cho, 10g/kg), iv) supplemented with egg phosphatidylcholine (PC) (PC, 10g/kg). On postnatal day (PD) 7, pups were culled to 4 from each dam, and kept on the same respective diets until 45 PD. On PD 35, memory function was measured by Morris water maze and on PD 45, retina function by an electroretinogram. Brain and retina were obtained for PL analysis by 31P NMR. Animals on the Def and PC diets were lower in body weights on PD 7, in comparison to the other two groups. While the Def group caught up in weights to its Cont counterparts, the PC group’s weight stayed consistently low until PD 45 (P<0.03). As for brain function, Cho and PC supplemented groups showed enhanced cued learning task, and spatial memory abilities, respectively, whereas the Def group showed the poorest memory recollection (P<0.05). The ERG amplitudes of rod driven photoreceptors and inner neural cell functions were significant (P<0.05) in the following order: Cont > Def > PC > Cho, at all light intensities, without reaching statistical significances in cone-driven responses. There were no differences in major PL compositions in the brain and retina. PC enriched group had increased subclasses of ether PL, PEaa and PCaa in the brain. These results indicate that while the addition of choline supplementation is beneficial for fetal brain development and function during early developmental stages, its contributions in the retina were minor. The effect of choline to the membrane PL structure was negligible for the stage of development in the given experimental design.

Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 42-43.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 18 (1958) no. 3, p. 778. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 1993. / Title from title page screen, viewed March 6, 2006. Dissertation Committee: Brian J. Wilkinson (chair), Herman E. Brockman, H. Tak Cheung, Radheshyam K. Jayaswal, Robert L. Preston. Includes bibliographical references (leaves 162-176) and abstract. Also available in print.

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 22-25). Also available via the World Wide Web.

No description available.

In response to salinity and drought, some higher plants accumulate the secondary metabolite glycinebetaine which functions as a compatible osmolyte (Rhodes and Hanson, 1993). Choline, a precursor of glycinebetaine (Rhodes and Hanson, 1993), is also a component of a primary metabolite phosphatidylcholine, an ubiquitous membrane phospholipid (Moore, 1982). In leaves of the glycinebetaine accumulator spinach, choline is synthesized as phosphocholine (PCho) and PCho is synthesized by three sequential N-methylations of phosphoethanolamine (PEA) ~ phosphomethyl-EA (PMEA) ~ phosphodimethyl-EA (PDEA) ~ PCho. The methyl group donor is Sadenosyl- L-methionine, SAM (Summers and Weretilnyk, 1993). The enzyme SAM: PMEA N-methyltransferase (PMEAMeT) is suggested to N-methylate PMEA ~ PDEA ~ PCho (Weretilnyk and Summers, 1992). A four-step strategy was developed for the partial purification of PMEAMeT on the basis of PMEA-dependent methylations (PMEAMeT activity) that involved the extraction of soluble leaf protein, ammonium sulfate precipitation, and column chromatography on DEAE Sepharose, Phenyl Sepharose and High Q Anion matrices. PDEA-dependent methylating activity co-purified with PMEAMeT activity which suggested PMEAMeT may N-methylate PMEA ~ PDEA ~ PCho. PMEAMeT was purified 43-fold and has specific activities of 14.7 and 18.0 nmol•min-1•mg-1 protein with PMEA and PDEA as substrates, respectively. Thin layer chromatography was used to identify the reaction products formed during the 30 minute assay incubation: with PMEA as the substrate, PDEA and PCho were detected in a ratio of 9: 1 as products; and with PDEA as the substrate, PCho was detected as the only product. PMEAMeT was estimated to have a native molecular mass of 76 kDa by HPLC gel filtration chromatography. Both PMEA and PDEA N-methylating activities have an alkaline pH optimum between 8.5 and 9.0 in 0.1 M Tris-HCl buffer. Neither activity was iii affected by the omission of Na2-EDT A from the assay. The addition of 10 mM Mg2+ to the assay inhibited PMEA and PDEA-dependent methylation by approximately 49% and 32%, respectively; whereas, the addition of 1 and 10 mM Mn2+ to the assay completely inhibited both activities. Both activities were inhibited by the reaction products S-adenosyl-Lhomocysteine by over 90% at 0.2 mM and PCho by approximately 80% at 10 mM. Of the products ofPCho hydrolysis, choline inhibited PMEA-dependent methylation by 10% at 10 mM; whereas, Pi inhibited PMEA and PDEA-dependent methylation by 38 and 19%, respectively at 10 mM. The compatible osmolyte glycinebetaine inhibited PMEA and PDEA-dependent methylation by between 20 and 30% at 140 mM; however, the inibition of PMEA-dependent methylation can be partly accounted for by the presence of cr ions in the assay. If present at these concentrations in the same subcellular compartment, these metabolites could serve as regulators ofPMEAMeT activities in vivo. Study of PMEAMeT contributed to identifying the number of enzymes that Nmethylate PEA ~ PMEA ~ PDEA ~ PCho and possible regulatory metabolites for choline biosynthesis in vivo. These data are pertinent to basic research and also to genetic-engineering studies aimed at introducing the glycinebetaine-accumulating trait into crop plants as an approach to enhancing osmotic-stress resistance. / Thesis / Master of Science (MSc)

Bibliography: leaves 194-219 / xxi, 219 leaves, [2] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1986

In the developing mouse oocyte, as well as in the preimplantation embryo, betaine (N,N,N-trimethylglycine) plays an important role first as a mechanism for cell volume regulation and second as a major methyl donor. Thus, the presence of betaine has implications both during development, and throughout the lifespan. It has previously been observed that betaine accumulates in the mouse oocyte as it matures, however its origin in the egg is unknown. Here I explore the enzyme choline dehydrogenase (CHDH; EC 1.199.1) as a method by which the mouse oocyte synthesizes the betaine that we observe prior to initiation transport activity in the preimplantation embryo. I carefully monitored betaine transport throughout meiotic maturation to confirm that no other previously unobserved membrane transport existed in the maturing oocyte. However, no betaine transport into oocytes was detected during meiotic maturation suggesting de novo synthesis. Previous data suggests that the enzyme is expressed (at the transcript level) in the developing oocyte, and becomes active during meiotic maturation. I demonstrated the presence of CHDH protein in the oocyte and preimplantation embryo. I then examined whether the mouse oocyte synthesizes betaine autonomously and addressed whether CHDH is a requirement for this process. Chdh knockout oocytes did not accumulate betaine in vivo, while normal betaine levels were observed in Chdh wildtype oocytes. CHDH-mediated synthesis of betaine was directly confirmed by detection of increased betaine in oocytes matured in vitro in the presence of choline. Chdh-/- oocytes failed to produce betaine when similarly cultured in choline. This establishes the production of betaine as an autonomous process in maturing oocytes. Overall, I have built upon previous data to demonstrate that betaine accumulation is a feature of meiotic maturation that occurs by de novo synthesis of the molecule, a process that requires transient activation of the enzyme choline dehydrogenase.

Choline acetyltransferase (ChAc), the enzyme responsible for the synthesis of acetylcholine (ACh), has been extensively purified from chicken brains. Purification procedures included ammonium sulfate fractionation, DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150 column chromatography, and affinity chromatography on agarose-hexane-Coenzyme A column. ChAc activity was measured radiochemically. Due to the instability of the enzyme in the course of purification, the most active fraction obtained after agarose-hexane-Coenzyme A chromatography showed a specific activity of only 0.34 μmoles ACh formed/ min./mg protein which corresponded to a 773 fold purification from homogenate. However, on non-denaturing polyacrylamide gel electrophoresis at pH 8.8, the highly purified ChAc preparation showed two distinct bands, and ChAc activity was recovered by slicing and assaying the gel. ChAc activity corresponded to the position of the faster moving band. The same preparation showed one major band and two minor bands on SDS gel electrophoresis. The estimated MW of chicken brain ChAc by gel filtration and SDS gel electrophoresis was 42,500 daltons and no subunit was observed. Two forms of chicken brain ChAc with different Km values for the substrates were eluted from agarose-hexane-CoA column. The pH optimum was estimated to be between pH 7.6-8.0. NaCl, KC1, Ca²⁺ and EDTA stimulated, while Cu²⁺, N-ethylmaleimide and CoA inhibited the enzyme preparation. The apparent Km values for acetyl-CoA and choline were, studied and were found to be similar to those of other mammalian species. The ChAc preparation also showed species specificity by the Ouchterlony double immuno diffusion test. Effect and mechanisms of salt and EDTA activation of ChAc activity are discussed. / Medicine, Faculty of / Graduate