Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 343-355).

N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the characteristic catalytic and regulatory domains. The expression of Ghpldδ1b showed hydrolytic and transphosphatidylation activity toward radiolabelled phosphatidylcholine (PC) but it appears Ghpldδ1b does not utilize NAPE as a substrate to produce NAEs nor does it seem to be suppressed by NAEs.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 20C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, their metabolism and functional implications in plants however, remain incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. FAAH orthologs, putative PpFAAHs (Physcomitrella patens FAAH) were identified based on the sequence blast of ratFAAH, and named as PpFAAH1 to PpFAAH10. Based on the highest mRNA expression of the PpFAAH homologs upon NAE treatment, PpFAAH1 was selected for further in vitro characterization, which shares 31% sequence identity with ratFAAH. PpFAAH1 was heterologously expressed in E. coli and purified for characterization. Highest amidohydrolysis activity of PpFAAH1 was observed in vitro at pH 8.0 and temperature 37°C. Methoxy arachidonyl fluorophosphonate (MAFP), an inhibitor showed highest inhibition with 10mM concentration, however, one of the principal classes of FAAH inhibitor O-aryl carbamates (URB597) exhibited only 22% inhibition with the same concentration. Both in vivo and in vitro studies showed that unsaturated NAE substrate (NAE 20:4) is hydrolyzed faster than the saturated NAE (NAE16:0); more than 50- and 10-fold higher in vitro and in vivo assays, respectively. Amidohydrolase activity in vivo was mostly associated with microsomes compared with cytoplasmic fractions. Additionally, microsomal fraction of mature gametophytes showed higher amidohydrolase activity than of the protonemal or early gametophyte stages; however, PpFAAH expression was not significantly different between the developmental stages. Further functional characterization of NAE metabolic pathway is ongoing by generation of PpFAAH knock out (KO) and overexpressor (OE) to understand the biological implications of FAAH in growth and development of early land plants.

Identification et caractérisation de la première N-acylphosphatidyléthanolamine synthase chez A. thaliana. Les N-acylphosphatidyléthanolamines (NAPE) sont des phospholipides complexes peu abondants au sein des membranes biologiques mais largement répandues dans différents organismes. Outre ses fonctions de stabilisation des membranes ce lipide est davantage connu pour être le précurseur des N-acyléthanolamines (NAE) qui sont impliquées dans de très nombreuses voies de signalisation chez les plantes (lors de la germination, du développement racinaire, de l’induction de gène de défense, etc.) comme chez les animaux (apoptose, ligand des récepteurs endocannabinoïdes, notion de satiété, etc.). Au début de ma thèse, les gènes codant pour les enzymes impliquées dans les différentes étapes de la voie métabolique des NAE (e.g NAPE-PLD, FAAH1 et 2) ont été caractérisées exceptées le ou les gène(s) codant pour l’enzyme catalysant la synthèse de NAPE précurseurs de ces lipides. Une étude bioinformatique a permis d’identifier de nouvelles séquences codantes pour des acyltransférases putatives chez A. thaliana dont celle du gène At1g78690. La caractérisation fonctionnelle de cette enzyme a été déterminée après son expression hétérologue chez E.coli sur fractions membranaires et protéines purifiées. Puis le profil d’expression génique, la localisation cellulaire de la protéine ainsi que son activité ont été étudiés chez les plantes à partir notamment de mutants d’A. thaliana (ADN-T et « 35S »). Les résultats obtenus au cours de cette étude ont permis d’identifier et de caractériser la première NAPE synthase chez les plantes. / Discovery and characterization of an A. thaliana N-acylphosphatidylethanolamine synthase. N-acylphosphatidylethanolamine (NAPE) is a widespread, albeit minor, membrane phospholipid in various organisms. Besides its stabilizing properties to membranes bilayers, NAPE is known to be the precursor for N-acylethanolamine (NAE) synthesis. NAE have been shown to regulate a variety of physiological functions in both plants (germination, root development, gene induction, etc.) and animals (apoptosis, ligand for cannabinoid receptors, satiety properties, etc.) At the beginning of my PhD, the genes encoding the enzymes involved in the different steps of NAE metabolism were well characterized (e.g NAPE-PLD, FAAH 1 and 2), with the exception of the NAPE synthase gene(s). A bioinformatic study allowed the identification of coding sequences for putative new acyltransferases in A. thaliana, such as the At1g78690 gene. After expression in E. coli, the functional characterization of At1g78690p was carried out by analyses of the lipid content and by enzymatic assays using membrane fractions or purified proteins. The localisation of the protein and its activity were also studied in A. thaliana mutants (ADN-T and “35S”). This study shows the identification and characterization of the first NAPE-synthase in plants.

N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro and in vivo for the molecular identification of Arabidopsis NAE amidohydrolase. Moreover, the plants with altered NAE amidohydrolase expression may provide new tools for improved understanding of the role of NAEs in germination and seedling growth.

This study was conducted to compare the gizzard integrity between two broilers flocks, one under a no antibiotics ever (NAE) program and the other from a conventionally raised flock (CONV). One-hundred Ross 708 x Ross YPM broiler chickens from two flocks of the same company but raised under two distinct management systems were allocated for this study. At 1, 14, 28 and 42 days of age, birds from both flocks were received at the Poultry Research and Diagnostic Laboratory. Gross and histopathologic evaluations of gizzard lesions and microbiological determinations of the gizzard mucosa were conducted and body and gizzard weights were recorded. Birds under both programs showed very similar growth rates, however gizzards were heavier in the birds under the CONV program. The birds under the NAE program had higher gross gizzard lesion scores at days 1, 14, 28 and 42, with statistical significance at day 42 (P<0.05). Histopathologically, the birds under the NAE program had greater gizzard lesion scores compared to the CONV flock at days 14 and 28, however no statistical significance was established. Escherichia coli was more frequently isolated from the birds under the CONV program. According to these observations, gross and microscopic gizzard lesions appeared to be more associated with the NAE program.

A neurite auto-imune experimental (NAE) é uma polineuropatia desmielinizante monofásica do sistema nervoso periférico (SNP). A NAE é considerada modelo experimental da síndrome de Guillain-Barré (SGB). Por se tratar de uma doença autoimune, pode ser induzida experimentalmente em camundongos geneticamente susceptíveis, através da imunização com componentes da mielina de nervos periféricos. Para a indução da NAE podem ser utilizados P0 e P2, proteínas da mielina do nervo periférico, ou sequências conhecidas de peptídeos dessas proteínas 180-199 e 58-81 respectivamente, consideradas neuritogênicas, ou ainda transferência adotiva de lifócitos T CD4+, oriundas de camundongos previamente imunizados. Para o presente estudo foram utilizados camundongos fêmeas SJL/J nãográvidas, com idade entre 8 e 12 semanas, pesando de 17 a 20 g. Os animais foram divididos em dois grupos: um controle e outro com NAE. 200 µg da sequência de peptídeos 58-81 de P2 emulsificada em 100 µl de adjuvante de Freund completo (AFC) foram injetados por via subcutânea, em quatro locais na região lombar. Para os controles, foi utilizado solução tamponada de fosfato (PBS), emulsionada em AFC desprovida da sequência 58-81 de peptídeos P2, injetada no mesmo local, na mesma quantidade e forma. Cada camundongo tratado com P2 recebeu 200 ng de toxina pertussis em 100 ?L de PBS intraperitonealmente (i.p.) nos dias 0 e 2 pós-imunização (p.i.). No grupo controle, volumes iguais de PBS e toxina pertussis foram administrados pela mesma via sem o peptídeo. Avaliações da motricidade foram realizadas diariamente até o 60º dia, além de análises funcionais e eletroneuromiográficas. Foram encontradas alterações exclusivamente eletrofisiológicas, desmielinizantes e axonais, em cerca de dois terços dos camundongos. Sendo o camundongo SJL/J considerado o camundongo mais susceptível para a provocação de NAE, os achados do presente estudo indicam a limitação do modelo: ausência de alterações motoras detectáveis clinicamente, ocorrendo distúrbios eletrofisiológicos em apenas parte dos animais. O melhor modelo de NAE continua sendo o provocado no rato Lewis por proteína da mielina periférica bovina. É desejável que se continue buscando modelo experimental de NAE em camundongos, tendo em vista que essa espécie animal é a mais bem estudada na Biologia animal e, por essa razão, dela haver extensa variedade de imunobiológicos disponíveis para estudo da patogenia e fisiopatologia de doenças auto-imunes. / Experimental autoimmune neurits (EAN) is a monophasic demyelinating disease of the peripheral nervous system (PNS). EAN is considered to be the experimental model for Guillain-Barré syndrome (GBS). EAN can be induced in genetically susceptible mice using peripheral myelin components as immunogens: peripheral nerve myelin proteins P0 or P2; peptides sequences of those proteins considered neuritogenic, or the adoptive transfer of TCD4+ lymphocytes from previously immunized mice. In the present study non-pregnant female SJL/J mice aged 8-12 weeks weighint 17-20 g where used. The experimental group was treated as follows. Peptides sequence of P2 (200 µg) emulsified in complete Freund adjuvant (CFA) (100 µl) injected in four sites at the lumbar paravertebral region s.c. Controls were injected in the same sites with equal quantities of phosphate buffer solution emulsified in CFA without the peptides sequence. Each mouse treated with P2 was also treated with 200 ng pertussis toxin in 100 ?L PBS i.p. at the days 0 and 2 post-immunisation. Controls were injected i.p. with equal volumes of PBS and pertussis toxin free of the peptides sequence. Motor strength, posture and coordination were evaluated daily until the 60th day postinoculation besides eletroneuromyographic (EMG) evaluation on the 10th and 30thy days of some mice. No clinical disturbances were observed and in two thirds of the animals demyelinating and axonal features were detected at the EMG. SJL/J mice are considered the most susceptible mouse strain for NAE induction but the findings of the present study indicates the limitations of the model and its reproducibility. The best NAE model is yet the obtained in Lewis rat. It is important to look for a mouse model of EAN because this animal species is the most studied in the animal Biology. The extensive variety of immunobiologic products from mice allows performing studies on the pathogeny or physiopathology of autoimmune diseases more easily.

A neurite auto-imune experimental (NAE) é uma polineuropatia desmielinizante monofásica do sistema nervoso periférico (SNP). A NAE é considerada modelo experimental da síndrome de Guillain-Barré (SGB). Por se tratar de uma doença autoimune, pode ser induzida experimentalmente em camundongos geneticamente susceptíveis, através da imunização com componentes da mielina de nervos periféricos. Para a indução da NAE podem ser utilizados P0 e P2, proteínas da mielina do nervo periférico, ou sequências conhecidas de peptídeos dessas proteínas 180-199 e 58-81 respectivamente, consideradas neuritogênicas, ou ainda transferência adotiva de lifócitos T CD4+, oriundas de camundongos previamente imunizados. Para o presente estudo foram utilizados camundongos fêmeas SJL/J nãográvidas, com idade entre 8 e 12 semanas, pesando de 17 a 20 g. Os animais foram divididos em dois grupos: um controle e outro com NAE. 200 µg da sequência de peptídeos 58-81 de P2 emulsificada em 100 µl de adjuvante de Freund completo (AFC) foram injetados por via subcutânea, em quatro locais na região lombar. Para os controles, foi utilizado solução tamponada de fosfato (PBS), emulsionada em AFC desprovida da sequência 58-81 de peptídeos P2, injetada no mesmo local, na mesma quantidade e forma. Cada camundongo tratado com P2 recebeu 200 ng de toxina pertussis em 100 ?L de PBS intraperitonealmente (i.p.) nos dias 0 e 2 pós-imunização (p.i.). No grupo controle, volumes iguais de PBS e toxina pertussis foram administrados pela mesma via sem o peptídeo. Avaliações da motricidade foram realizadas diariamente até o 60º dia, além de análises funcionais e eletroneuromiográficas. Foram encontradas alterações exclusivamente eletrofisiológicas, desmielinizantes e axonais, em cerca de dois terços dos camundongos. Sendo o camundongo SJL/J considerado o camundongo mais susceptível para a provocação de NAE, os achados do presente estudo indicam a limitação do modelo: ausência de alterações motoras detectáveis clinicamente, ocorrendo distúrbios eletrofisiológicos em apenas parte dos animais. O melhor modelo de NAE continua sendo o provocado no rato Lewis por proteína da mielina periférica bovina. É desejável que se continue buscando modelo experimental de NAE em camundongos, tendo em vista que essa espécie animal é a mais bem estudada na Biologia animal e, por essa razão, dela haver extensa variedade de imunobiológicos disponíveis para estudo da patogenia e fisiopatologia de doenças auto-imunes. / Experimental autoimmune neurits (EAN) is a monophasic demyelinating disease of the peripheral nervous system (PNS). EAN is considered to be the experimental model for Guillain-Barré syndrome (GBS). EAN can be induced in genetically susceptible mice using peripheral myelin components as immunogens: peripheral nerve myelin proteins P0 or P2; peptides sequences of those proteins considered neuritogenic, or the adoptive transfer of TCD4+ lymphocytes from previously immunized mice. In the present study non-pregnant female SJL/J mice aged 8-12 weeks weighint 17-20 g where used. The experimental group was treated as follows. Peptides sequence of P2 (200 µg) emulsified in complete Freund adjuvant (CFA) (100 µl) injected in four sites at the lumbar paravertebral region s.c. Controls were injected in the same sites with equal quantities of phosphate buffer solution emulsified in CFA without the peptides sequence. Each mouse treated with P2 was also treated with 200 ng pertussis toxin in 100 ?L PBS i.p. at the days 0 and 2 post-immunisation. Controls were injected i.p. with equal volumes of PBS and pertussis toxin free of the peptides sequence. Motor strength, posture and coordination were evaluated daily until the 60th day postinoculation besides eletroneuromyographic (EMG) evaluation on the 10th and 30thy days of some mice. No clinical disturbances were observed and in two thirds of the animals demyelinating and axonal features were detected at the EMG. SJL/J mice are considered the most susceptible mouse strain for NAE induction but the findings of the present study indicates the limitations of the model and its reproducibility. The best NAE model is yet the obtained in Lewis rat. It is important to look for a mouse model of EAN because this animal species is the most studied in the animal Biology. The extensive variety of immunobiologic products from mice allows performing studies on the pathogeny or physiopathology of autoimmune diseases more easily.

N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. The abi3-1 mutant showed no expression of ABI3 and AtHVA22B, but RD29B expression remained similar to wildtype seedlings, suggesting an alternate mechanism for NAE and ABA interaction. Taken together, these data suggest that NAE metabolism acts through ABI3-dependent and independent pathways in the negative regulation of seedling development.

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