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The postnatal development of rat retinal phenylethanolamine-N-methyltransferase (PNMT) activity was measured by radiometric assay. Activity was detected on day 1 of life. Retinal PNMT activity of day 1 neonates approximated 10% that of the adult. There is an increase in enzyme activity before eye opening. By day 30, enzyme activity has peaked. The enzyme during this early period possesses the same substrate specificity and inhibitor sensitivity as that of the adult enzyme. PNMT activity is detected before tyrosine hydroxylase activity.

To identify the signaling mechanisms and components that are involved in regulation of a promoter for a gene involved in a secondary pathway I studied the nicotinic alkaloid biosynthetic pathway using various N. tabacum tissues. Nicotine and tropane alkaloids are widely known to be synthesized predominantly in the roots of species that produce pyrrolinium ring containing alkaloids. Putrescine Nmethyltransferase (PMT) catalyzes the first committed step in the biosynthesis of these alkaloid secondary products and earlier studies have indicated that PMT gene expression is restricted to root tissue in Solanaceae plants. To further elucidate the factors that govern the regulation of alkaloid synthesis, expression patterns dictated by the 5'-flanking region of one of the members of the PMT -gene family, NsPMT3, using the b-glucuronidase (GUS) reporter gene were examined. Various treatments were used to characterize the nature of signaling in various tissues of seedlings, whole plants and callus. High expression levels were detected in root tissue and no expression was detected in leaves, in agreement with previous studies. However, mechanically wounded leaves resulted in highly localized PMT expression. This wound-induced expression was transient, with maximum levels occurring immediately after wounding and diminishing after approximately 24 h. RT-PCR analysis of mRNA isolated from wild-type plants also indicated upregulation of PMT expression in leaves upon wounding as well as very low transcript levels in unwounded leaves. Low levels of PMT activity were detected in leaf tissue, and this activity did not increase significantly upon wounding. Transgenic callus material showed strong repression of PMT promoter activity in the presence of light and auxin, whereas dark conditions and the absence of auxin upregulated PMT promoter activity. Reactive oxygen species have been implicated in signaling. When treated with the scavengers of reactive oxygen species (ROS), dimethylthiourea (DMTU) or catalase, tobacco callus tissue, which displays highly repressed alkaloid synthesis under normal culture conditions in the light, exhibited significant induction of PMT promoter activity and alkaloid accumulation. It is thought that light repression signals through an ROS intermediate to affect changes in alkaloid pathway gene expression. Upregulation of PMT-promoter activity was observed upon treatment with JA (jasmonic acid) or darkness in roots of very young transgenic seedlings. Treatment with auxin, salicylic acid (SA) and H2O2, on the other hand, was found to highly repress PMT promoter activity. Action of other ROS such as nitric oxide and superoxide radicals on PMT expression is not clear but probably play less of a role, compared to H2O2. Consistent with this content ion, treatment with light or glucose oxidase (GOX) and glucose to generate H2O2, also repressed alkaloid accumulation, and treatment of seedlings to dark conditions, the ROS scavenger DMTU, or jasmonic acid resulted in alkaloid accumulation. Long distance signaling from leaves to roots is also suspected to involve ROS, as leaves treated with GOX and glucose exhibited repressed PMT promoter activity in roots. The responses of the PMT promoter to auxin, salicylic acid and H2O2 treatments were conserved as sho wn by similar responses of the N. tabacum PMT promoter when examined in transgenic Arabidopsis, thereby suggesting that these molecules signal through a conserved mechanism. Thus, ROS is strongly implicated in acting as an intermediate in these signaling processes with H2O2 proposed as a major signaling component.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24516号 / 医博第4958号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 岩田 想, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p> In plants, phosphocholine (PCho) is a precursor to the membrane component phosphatidylcholine (PtdCho) and free choline (Cho). A mutant Saccharomyces cerevisiae yeast strain unable to produce PtdCho without exogenous choline was used for transformation with an Arabidopsis cDNA library cloned in the yeast expression vector pFK61. A plant cDNA associated with locus At1g48600 functionally complemented the mutant by restoring growth on minimal synthetic medium lacking choline but containing the phosphobase phosphomethylethanolamine (PMEA). Crude extracts prepared from the yeast showed a novel capacity to convert PMEA to phosphodimethylethanolamine (PDEA) and PCho and hence this enzyme has been named Arabidopsis S-adenosyl-L-methionine (AdoMet): phosphomethylethanolamine N-methyltransferase (AtPMEAMT). </p> <p> AtPMEAMT is a bipartite enzyme containing tandem N-and C-terminal AdoMet-binding domains. The predicted amino acid sequence shows an 87% identity to the previously characterized AdoMet: phosphoethanolamine N-methyltransferase (AtPEAMT) from Arabidopsis. An important distinction between AtPMEAMT and AtPEAMT is that the former enzyme is unable to methylate phosphoethanolamine (PEA). However, both AtPEAMT and AtPMEAMT can methylate PMEA and PDEA, two phosphobase intermediates ofPCho synthesis. The apparent Km values were determined for AtPEAMT and AtPMEAMT toward PMEA and PDEA and found to be 0.32 and 0.14 mM, respectively, for PEAMT and 0.16 and 0.03 mM, respectively, for PMEAMT. The N-and C-terminal Ado Met-domains of PEAMT and PMEAMT were cloned separately into a pET30a(+) vector for protein expression and extracts containing recombinant proteins were assayed for phosphobase methyltransferase activity. Only the gene product encoding the domain associated with the C-terminal half of PMEAMT methylated both PMEA and PDEA, an activity found with the native protein. A chimera was produced by combining the N-terminal half ofPEAMT and the C-terminal half of PMEAMT. The chimeric protein is able to methylate PEA, PMEA and PDEA indicating that a feature associated with the N-terminal half of PEAMT is required for PEA methylation. This result suggests that differences associated with the N-terminal domain are likely responsible for the inability ofPMEAMT to use PEA as a substrate. </p> <p> An Arabidopsis mutant line with a T-DNA insertion in the promoter region of PMEAMT (SALK 006037) was obtained and RT-PCR analysis of plants homozygous for the insert showed that the mutant lacks transcripts associated with this gene. Relative to wild-type plants grown under identical conditions the mutant plants showed no visible difference in morphological or developmental phenotype. However, shotgun lipidomics using electrospray ionization tandem mass spectrometry showed a 2.1-fold greater abundance ofa 34:3 phosphatidylmethylethanolamine (PtdMEA) molecular species in mutant plants compared to wild-type. One biological role of PMEAMT may be to reduce the likelihood for PtdMEA incorporation into phospholipids ofmembranes. PtdMEA incorporation in membranes is associated with reduced viability of yeast but its effect on the physiology ofplants is, as yet, unknown. </p> / Thesis / Doctor of Philosophy (PhD)

<p> Some plants such as spinach, sugar beet, and wheat accumulate the quaternary ammonium compound glycine betaine when exposed to stresses in their environment. Environmental stress can be in the form of an excess or deficiency of water, high salt content, and/or exposure to excessively low or high temperatures and many if not all of these stresses are associated with cell dehydration. </p> <p> Glycine betaine is an organic solute that is believed to help restore the osmotic potential of a cell undergoing dehydration by reducing water loss and preventing damage to the structure and function of macromolecules. However, many plants such as Arabidopsis, tobacco, and rice do not accumulate glycine betaine. Given the perceived benefits of glycine betaine production by plants under stress, studies have been carried out to identify factors regulating its production. </p> <p> Glycine betaine is synthesized by the two-step oxidation of choline. The capacity to synthesize phosphocholine for choline production has been found to limit the production of glycine betaine in non-accumulating plants such as tobacco. As such, genetic engineering has been used to enhance the production of choline to up-regulate the synthesis of glycine betaine. This strategy has required knowledge of the enzyme(s) catalyzing the three N-methylation steps of the phosphocholine biosynthetic pathway. </p> <p> This study focused on a gene product identified as putative-phosphoethanolamine N-methyltransferase (putative PEAMT) based upon its similarity to a spinach Nmethyltransferase known to convert phosphoethanolamine to phosphocholine. This gene is located at the locus Atlg73600 on chromosome I of Arabidopsis and its predicted amino acid sequence has high similarity to two other genes encoding N-methylating enzymes located at At3 g 18000 (a biochemically confirmed PEAMT) and At 1 g48600 (annotated as a putative PEAMT). </p> <p> In this study, publicly available microarray data was examined to identify an expression profile of transcripts associated with the Atlg73600 gene in organs and tissues of Arabidopsis at various developmental stages. A summary of the micro array data shows the highest abundance of transcripts for Atlg73600 to be in the rosette leaves of Arabidopsis at 18.0- 20.9 days of growth. </p> <p> Arabidopsis plants grown from seeds from four SALK lines reported to have a TDNA insert in the Atlg73600 gene were screened for the presence of a T-DNA tag using a three primer PCR design strategy. Individual plants from two of the lines were found to have a T-DNA insert present. RT-PCR was then used to analyze the expression of transcripts associated with the Atlg73600 gene in these mutant lines. Transcripts were not detected among the amplified products from eDNA produced from the SALK line designated 062703 but they were found at reduced levels in eDNA of SALK line 016929c. </p> <p> In future studies the two T -DNA mutant lines identified in this study can be used to assign a biological role for the product of the Atlg73600 gene by examining the phenotype of these mutant plants relative to that of wild-type plants under normal and/or stressed conditions. The line found with no expression associated with the Atlg73600 gene will be useful in crosses with T -DNA knock-out mutants of genes at loci At3g18000 and Atlg48600. Systematic knock-outs for each of the genes in isolation and in combination will help discern whether there is functional redundancy in their biological roles or if their individual expression contributes uniquely towards the development of a plant or its stress response. Given the associated role for PEAMT in phosphatidylcholine metabolism, lipidomics could be used to determine if the composition of the plant membranes is altered relative to wild-type when the Atlg73600 gene is knocked-out. </p> / Thesis / Master of Science (MSc)

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF. Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.

Plants produce a vast number of low-molecular-weight chemicals (so called secondary or specialized metabolites) that confer a selective advantage to the plant, such as defense against herbivory or protection from changing environmental conditions. Many of these specialized metabolites are used for their medicinal properties, as lead compounds in drug discovery, or to impart our food with different tastes and scents. These chemicals are produced by various pathways of enzyme-mediated reactions in plant cells. It is suspected that enzymes in plant specialized metabolism evolved from those in primary metabolism. Understanding how plants evolved to produce these diverse metabolites is of primary interest, as it can lead to the engineering of plants to be more resistant to both biotic and abiotic stress, or to produce more complex small molecule compounds that are difficult to derive. To that end, the first objective was to develop a schema for rational protein engineering using meta-analyses of a well-characterized sesquiterpene synthase family encoding two closely-related but different types of enzymes, using quantitative measures of natural selection on amino-acid positions previously demonstrated as important for neofunctionalization between two terpene synthase gene families. The change in the nonsynonymous to synonymous mutation rate ratio (dN/dS) between these two gene families was large at the sites known to be responsible for interconversion. This led to a metric (delta dN/dS) that might have some predictive power. This natural selection-oriented approach was tested on two related enzyme families involved in either nicotine/tropane alkaloid biosynthesis (putrescine N-methyltransferase) or primary metabolism (spermidine synthase) by attempting to interconvert a spermidine synthase to encode putrescine N-methyltransferase activity based upon past patterns of natural selection. In contrast to the HPS/TEAS system, using delta dN/dS metrics between SPDS and PMT and site directed mutagenesis of SPDS did not result in the desired neofunctionalization to PMT activity. Phylogenetic analyses were performed to investigate the molecular evolution of plant N-methyltransferases involved in three alkaloid biosynthetic pathways. The results from these studies indicated that unlike O-MTs that show monophyletic origins, plant N-MTs showed patterns indicating polyphyletic origins. To provide the foundation for future molecular-oriented studies of urushiol production in poison ivy, the complete poison ivy root and leaf transcriptomes were sequenced, assembled, and analyzed. / Ph. D.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways. / QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA. / Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes. / Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.