Efeito protetor da acetamida sobre as intoxicações experimentais em ratos por monofluoroacetato de sódio e por algumas plantas brasileiras que causam morte súbita

O objetivo deste trabalho é avaliar e comparar o efeito protetor da acetamida nas intoxicações por monofluoroacetato de sódio (MF) e por oito plantas brasileiras que causam "morte súbita" (PBCMS) (Palicourea marcgravii, P. juruana, Pseudocalymma elegans, Arrabidaea bilabiata, Amorimia (Mascagnia) rigida, M. pubiflora, Amorimia (Mascagnia) exotropica e M. aff. rigida) em ratos, bem como descrever o quadro clínico-patológico nos animais intoxicados. Foram utilizados 33 ratos da linhagem Wistar (Rattus norvegicus albinus), nove dos quais ingeriram espontaneamente folhas frescas de P. marcgravii nas doses de 2,0 e 4,0g/kg. Dois desses ratos receberam doses únicas de acetamida de 2,0 e 4,0g/kg, por via oral um minuto antes do fornecimento da planta. Outro rato recebeu 4,0g/kg de acetamida após ingerir 4,0g/kg da planta e manifestar sintomas graves. Nos experimentos com MF, foram administradas a quatro ratos doses de 4,0 e 8,0mg/kg; o intervalo de tempo entre a administração da acetamida e dos extratos concentrados ou do MF variou entre 2 a 4 horas. A dose de acetamida utilizada variou de 2,0 a 8,0g/kg. Nos experimentos com extratos concentrados das oito PBCMS, 20 ratos foram intoxicados por via oral com doses únicas ou repetidas. A acetamida, quando previamente administrada em doses suficientemente altas, evitou o aparecimento dos sinais clínicos ou morte dos animais intoxicados por MF, bem como pelas folhas frescas de P. marcgravii e com os extratos concentrados de cada uma das sete outras PBCMS utilizadas. Todos esses ratos foram novamente submetidos ao mesmo protocolo experimental, porém sem administração de acetamida. Nesses experimentos posteriores todos os ratos manifestaram sinais clínicos e morte (exceto M. aff. rigida, cuja amostra não se revelou tóxica). Todos os ratos apresentaram aurículas de leve a acentuadamente ingurgitadas e, por vezes, também as veias cava cranial e caudal. Havia moderada dilatação cardíaca direita e esquerda em três animais. O fígado de todos os animais apresentava-se de leve a acentuadamente congesto, e em alguns ratos, verificou-se evidenciação do padrão lobular. Observou-se ainda discreta a leve presença de líquido espumoso na superfície de corte dos pulmões em três ratos. O exame histopatológico evidenciou nos rins de seis dos 30 ratos leve a moderada tumefação citoplasmática dos túbulos uriníferos contornados distais e, por vezes, também nos túbulos coletores, mas somente em quatro (dois intoxicados por P. marcgravii, um por P. elegans e outro por A. exotropica) havia a clássica lesão de degeneração hidrópico-vacuolar com picnose nuclear evidente. No fígado de 26 animais havia congestão e, destes, três apresentaram estreitamento compressivo dos cordões hepáticos, oito corpúsculos de choque; em outros três, necrose focal de discreta a moderada. Observou-se ainda, de discreta a moderada vacuolização de hepatócitos em 16 animais. Este trabalho demonstra que a acetamida possui acentuado efeito protetor, capaz de prevenir os sinais clínicos e evitar a morte dos ratos intoxicados por MF, folhas frescas de P. marcgravii e extratos concentrados de outras sete PBCMS. O quadro clínico-patológico observado nos ratos intoxicados pelo MF e pelas PBCMS deste estudo, associado ao efeito protetor da acetamida, confirma que o MF é o princípio tóxico responsável pela morte dos ratos, e, por extensão, também dos bovinos que ingeriram PBCMS.

Fungos zoospóricos (Mastigomycotina) da mata atlântica da Reserva Biológica do Alto da Serra de Paranapiacaba, município de Santo André, SP

Trezentas e dezesseis ocorrências de 20 táxons de Mastigomycotina (fungos zoospóricos) foram registradas em folhas de Alchornea triplinervia (Spreng.) Müll. Arg. colocadas em ambientes terrestre e aquático, em amostras de solo e de água de riacho coletadas mensalmente de julho de 1988 a maio de 1990. Entre os 13 táxons de Chytridiomycetes e sete de Oomycetes, as espécies com maior ocorrência foram: Karlingia rosea (De Bary & Woronin) Johanson (35 ocorrências), Polychytrium aggregatum Ajello (32 ocorrências), Rhizophydium elyensis Sparrow (34 ocorrências) e Nowakowskiella elegans (Nowak.) Schroeter (32 ocorrências). São citados pela primeira vez para a mata atlântica: Karlingiomyces sp., Phlyctochytrium sp. e Rhizophydium chitinophyllum Sparrow.

Morfo-anatomia do embrião de leguminosas arbóreas nativas

Objetivando caracterizar os eixos embrionários de sementes de espécies de Fabaceae e fornecer subsídios para trabalhos de sistemática e filogenia desta família, foram estudados aspectos morfológicos e anatômicos dos embriões, especialmente dos eixos embrionários, de 15 espécies arbóreas nativas, buscando correlações entre sua estrutura e a afinidade entre os diferentes gêneros e destes nas tribos. Observaram-se cotilédones foliáceos nas espécies de Caesalpinioideae, carnosos nas Faboideae e dos dois tipos nas Mimosoideae. Os eixos embrionários variaram de curtos a longos, sendo sempre retos nas espécies de Caesalpinioideae e Mimosoideae; nas Faboideae, encontraram-se eixos embrionários curvos ou inclinados. A plúmula variou de indiferenciada a diferenciada, o primeiro tipo ocorrendo em espécies sem epicótilo e o último disposto sobre epicótilo alongado. Anatomicamente, a protoderme se mostrou indiferenciada na maioria das espécies, podendo ocorrer tricomas em diferenciação (Caesalpinia leiostachya, Centrolobium tomentosum e Anadenanthera macrocarpa), papilas (Platypodium elegans) e tricomas tectores e glandulares diferenciados (Inga urugüensis). O procâmbio se manteve indiferenciado. O meristema fundamental mostrou acúmulo de amido em todas as espécies, ocorrendo drusas em Peltophorum dubium e cristais poliédricos isolados em Inga urugüensis e em Lonchocarpus muehlbergianus. Esta última espécie apresentou, ainda, estruturas secretoras na região do nó cotiledonar.

Temporal dynamics of two species of Chaetophoraceae (Chlorophyta) in tropical streams of São Paulo State, Southeastern Brazil

Temporal dynamics of the chaetophoracean green algae Chaetophora elegans (Roth) C.A. Agardh and Stigeoclonium amoenum Kützing populations was investigated biweekly during late autumn trhough early spring (April to October) in two tropical streams from northwestern São Paulo State, southeastern Brazil. Abundances of one population of each species was evaluated by the quadrat technique in terms of percent cover and frequency. The fluctuations were related to the following stream variables: temperature, turbidity, specific conductance, pH, oxygen saturation, depth, substratum type, current velocity, irradiance and nutrients. Percent cover and frequency of C. elegans had lower values throughout the study period and was positively correlated to rainfall. Other correlations (i.e. positive of percent cover with depth and current velocity and negative with irradiance) were consistently found, reinforcing the strong influence of rainfall. On the other hand, percent cover and frequency of S. amoenum had higher values, with maximum growth from June to September. Percent cover was negatively correlated to rainfall. Results suggest the precipitation regime as the most important driving force to temporal changes in both populations, but playing different roles in each one. The gelatinous thallus of C. elegans seem to be favored by the increment of current velocity, since higher flows can improve the nutrient uptake by means of reduction in diffusion shell without promoting excessive drag force. In contrast, tufts of S. amoenum are, presumably, more exposed to drag force, and, consequently, more susceptible to mechanical damage effects due to higher current velocities.

Fisionomia e estrutura de uma floresta estacional montana do maciço da Borborema, Pernambuco - Brasil

Foi caracterizada a fisionomia e a estrutura do componente arbóreo de um fragmento de floresta estacional montana situado a 900 m de altitude. A área de estudo (8º11'144" -8º12'27" S e 36º23'730" -36º24'638" W) apresenta solos profundos e precipitação média anual de 948 mm ano-1. Foram amostrados todos os indivíduos vivos ou mortos, ainda em pé, com diâmetro a altura do peito > 5 cm, em 50 parcelas contíguas de 10 x 20 m, e tomadas as seguintes medidas: altura total, diâmetro do caule e área de cobertura da copa. As espécies foram classificadas quanto ao tamanho foliar, deciduidade e composição do limbo. Um total de 62 espécies (um taxon não identificado) foi amostrado. Densidade total, área basal total, altura e diâmetro médios e máximos dos indivíduos vivos foram 1.553 ind ha-1, 39 m² ha-1, 10,3 e 30 m e 14,2 e 105,0 cm, respectivamente. Cerca de 50% dos indivíduos vivos ocorreram abaixo de 10 m de altura. A distribuição das áreas basais e cobertura de copa por classe de altura indicou dois intervalos de concentração: 7-13 e 19-22 m. A área representa uma floresta estacional montana de transição entre as florestas ombrófilas e estacionais. A maioria das espécies é perenifólia e apresenta folhas simples e micrófilas (Casearia sylvestris Sw., Guapira nitida (Schmidt) Lundell, Marlierea clausseniana (O. Berg) Kiaersk., Ocotea aff. elegans Mez, Plinia sp., entre outras), ocupando o dossel da floresta juntamente com espécies notófilas (Amaioua cf. guianensis Aubl. e Roupala paulensis Sleumer) e mesófilas (Fabaceae sp. e Inga marginata Willd., entre outras). Entre as emergentes predominam espécies com folhas caducifólias e compostas, como Copaifera trapezifolia Hayne e Eriotheca crenulaticalyx A. Robyns.

Aquatic Hyphomycetes in the Parque Estadual das Fontes do Ipiranga - PEFI, São Paulo, Brazil

In the State Park Parque Estadual das Fontes do Ipiranga - PEFI, located in the city of São Paulo, samples of submerged mixed leaf litter were collected monthly from 10 sites with different levels of eutrophication, from October 2003 to April 2005. Some abiotic factors, such as pH, temperature, conductivity and dissolved oxygen were simultaneously measured in 20 cm deep water at each site. The leaf litter samples were washed in laboratory, cut into 1 cm² pieces and incubated in Petri dishes containing distilled sterile water for five to seven days at 15 °C to 20 °C. Twenty-four aquatic Hyphomycete species were identified, with predominance of Anguillospora crassa Ingold, Lunulospora curvula Ingold, Tetrachaetum elegans Ingold and Camposporium pellucidum (Grove) Hughes. First reports for South America are: Anguillospora filiformis Greathead, Dendrospora erecta Ingold and Pyramidospora casuarinae Nilsson. These species, as well as Tetracladium setigerum (Grove) Ingold, Tricladium splendens Ingold and Varicosporium elodeae Kegel, are reported for the first time in Brazil. According to the multivariate analysis, the occurrence of the aquatic Hyphomycetes was mainly influenced by the trophic level of the aquatic environments.

Neutralization of the edema-forming, defibrinating and coagulant effects of Bothrops asper venom by extracts of plants used by healers in Colombia

We determined the neutralizing activity of 12 ethanolic extracts of plants against the edema-forming, defibrinating and coagulant effects of Bothrops asper venom in Swiss Webster mice. The material used consisted of the leaves and branches of Bixa orellana (Bixaceae), Ficus nymphaeifolia (Moraceae), Struthanthus orbicularis (Loranthaceae) and Gonzalagunia panamensis (Rubiaceae); the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); the whole plant of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); rhizomes of Renealmia alpinia (Zingiberaceae), Heliconia curtispatha (Heliconiaceae) and Dracontium croatii (Araceae), and the ripe fruit of Citrus limon (Rutaceae). After preincubation of varying amounts of each extract with either 1.0 µg venom for the edema-forming effect or 2.0 µg venom for the defibrinating effect, the mixture was injected subcutaneously (sc) into the right foot pad or intravenously into the tail, respectively, to groups of four mice (18-20 g). All extracts (6.2-200 µg/mouse) partially neutralized the edema-forming activity of venom in a dose-dependent manner (58-76% inhibition), with B. orellana, S. orbicularis, G. panamensis, B. rosademonte, and D. croatii showing the highest effect. Ten extracts (3.9-2000 µg/mouse) also showed 100% neutralizing ability against the defibrinating effect of venom, and nine prolonged the coagulation time induced by the venom. When the extracts were administered either before or after venom injection, the neutralization of the edema-forming effect was lower than 40% for all extracts, and none of them neutralized the defibrinating effect of venom. When they were administered in situ (sc at the same site 5 min after venom injection), the neutralization of edema increased for six extracts, reaching levels up to 64% for C. limon.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

Antiprotozoal and molluscicidal activities of five Brazilian plants

Leishmaniasis, Chagas' disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people. In the present study, biological assays for antiprotozoal and molluscicidal activities were carried out with ethanolic extracts of plant species from the Brazilian part of the Upper Paraná River. Crude extracts were obtained by percolation with absolute ethanol from the leaves of Cayaponia podantha Cogn., Nectandra falcifolia (Nees) Castiglioni and Paullinia elegans Cambess., as well as from the aerial parts of Helicteres gardneriana St. Hil. & Naud. and Melochia arenosa Benth., all belonging to genera used in folk medicine. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose. Anti-leishmanial activity was determined over cultures of promastigote forms of the parasite in Schneider's Drosophila medium. Microscopic countings of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the percentage of growth inhibition. C. podantha and M. arenosa, at a concentration of 10 µg/mL, showed 90.4 ± 11.52 and 88.9 ± 2.20% growth inhibition, respectively, of epimastigote forms of Trypanosoma cruzi, whereas N. falcifolia demonstrated an LD50 of 138.5 µg/mL against promastigote forms of Leishmania (Viannia) braziliensis. Regarding molluscicidal activity, the acute toxicity of the extracts on Biomphalaria glabrata was evaluated by a rapid screening procedure. M. arenosa was 100% lethal to snails at 200 µg/mL and showed an LD50 of 143 µg/mL. Screening of plant extracts represents a continuous effort to find new antiparasitic drugs.

Identification of a new human mtDNA polymorphism (A14290G) in the NADH dehydrogenase subunit 6 gene

Leber's hereditary optic neuropathy (LHON) is a maternally inherited form of retinal ganglion cell degeneration leading to optic atrophy in young adults. Several mutations in different genes can cause LHON (heterogeneity). The ND6 gene is one of the mitochondrial genes that encodes subunit 6 of complex I of the respiratory chain. This gene is a hot spot gene. Fourteen Persian LHON patients were analyzed with single-strand conformational polymorphism and DNA sequencing techniques. None of these patients had four primary mutations, G3460A, G11788A, T14484C, and G14459A, related to this disease. We identified twelve nucleotide substitutions, G13702C, T13879C, T14110C, C14167T, G14199T, A14233G, G14272C, A14290G, G14365C, G14368C, T14766C, and T14798C. Eleven of twelve nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A14290G) has not been reported. The A14290G nucleotide substitution does not change its amino acid (glutamic acid). We looked for base conservation using DNA star software (MEGALIGN program) as a criterion for pathogenic or nonpathogenic nucleotide substitution in A14290G. The results of ND6 gene alignment in humans and in other species (mouse, cow, elegans worm, and Neurospora crassa mold) revealed that the 14290th base was not conserved. Fifty normal controls were also investigated for this polymorphism in the Iranian population and two had A14290G polymorphism (4%). This study provides evidence that the mtDNA A14290G allele is a new nonpathogenic polymorphism. We suggest follow-up studies regarding this polymorphism in different populations.

Nucleotide-sugar transporters: structure, function and roles in vivo

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

Fungos em sementes de plantas ornamentais

Estudou-se a ocorrência de fungos em sementes de nove espécies de plantas ornamentais herbáceas (Dahlia pinnata, Petunia x hybrida, Phlox drummondii, Rudbeckia hirta, Salvia farinacea, Salvia splendens, Tagetes patula, Viola tricolor e Zinnia elegans) costumeiramente plantadas no Distrito Federal. O método de detecção utilizado foi o de papel de filtro ("blotter-test"), sendo que uma subamostra, de 100 sementes, de cada espécie foi submetida a assepsia com álcool 70% e hipoclorito de sódio 1% e outra não. Das amostras de sementes analisadas foram detectados e isolados 32 fungos, sendo 88% representantes do grupo dos fungos mitospóricos, 6% do filo Ascomycota, 3% do filo Zygomycota e 3% de organismos semelhantes à fungos do filo Oomycota. Os gêneros mais frequentemente encontrados foram Alternaria, Cladosporium, Bipolaris, Curvularia, Exerohilum, Aspergillus e Penicillium. O maior número de fungos ocorreu nas sementes de D. pinnata, T. patula e P. drummondii. Alternaria alternata, Alternaria spp, Bipolaris spp., Curvularia lunata, C. protuberata, Exserohilum sp., Phoma glomerata, P. multirostrata, Pythium sp. e Ulocladium atrum podem, por indícios literários, ser um relato pioneiro em algumas das plantas de ornamentais. Possivelmente, no Brasil este é o primeiro relato de: Alternaria alternata em sementes de Dahlia pinnata, Salvia farinacea, S. splendens e Tagetes patula; Curvularia lunata em sementes de Rudbeckia hirta e T. patula; C. protuberata ePhoma glomerata em Zinnia elegans; Phoma multirostrata em Salvia splendens, e; Ulocladium atrum em semente de Viola tricolor.

Biotransformation of polycylic aromatic compounds by fungi and an investigation into the oxidation of alkylbenzenes by Mortierella isabellina NRRL 1757

Incubations of several polycyclic heteroaromatic compounds and two polycyclic aromatic hydrocarbons with a series of common fungi have been performed. The fungi Cunninghamella elegans ATCC 26269, Rhizopus arrhizus ATCC 11145, and Mortierella isabellina NRRL 1757 were studied in this regard. Of the aza heteroaromatics, only dibenzopyrrole gave a ring hydroxylated product following the incubation with C. elegans. From the thio heteroaromatics studied, dibenzothiophene was metabolized by all the three fungi and thioxanthone by C. elegans and M. isabellina giving sulfones and sulphoxides. Thiochromanone was metabolized stereoselectively to the corresponding sulphoxide by C. elegans. Methyl substituted thioxanthones on incubation with C. elegans produced oxidative products, arising from S -oxidation and hydroxylation at the methyl group. Of the cyclic ketones studied, only fluorenone was reduced to hydroxyfluorene and this metabolism is compared with that reported with cytochrome P-450 monooxygenases of hepatic microsomes. A series of para-substituted ethylbenzenes has been transformed stereoselectively to the 1-phenylethanols by incubation with M. isabellina. Comparisons of the enantiomeric purities obtained from products with their respective para substituent of the same steric size but different electronic properties indicate that the stereoselectivity of hydroxylation at benzylic carbon may be susceptible to electron donating or withdrawing factors in some cases, but that observation is not va lid in all the comparisons. The stereochemistry of the reaction is discussed in terms of three possible steps, ethylbenzene ---) 1-phenylethanol ---) acetophenone ---) 1-phenylethanol. This metabolic pathway could account for the inconsistencies observed in the comparisons of optical purities and electronic character of para substituents. Furthermore, formation of 2-phenylethanol (in some cases), l-(p-acetylphenyl)ethanol from p-diethylbenzene, and N-acetylation of p-ethylaniline was observed. n-Propylbenzene was also converted to optically active 1-phenylpropanol. Acetophenone, p-ethylacetophenone, and o(,~,~-trifluoroacetophenone were transformed to 1-phenylethanol, l-(p-ethylphenyl)ethanol, and 1-phenyl-2,2,2-trifluoroethanol, respectively, with high chemical and excellent optical yields. The 13 C NMR spectra of several substrates and metabolic products have been reported and assigned for the first time.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.