Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst

<p>Abstract</p> <p>Background</p> <p>Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity. Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6β-hydroxyhyoscyamine) is the intermediate in the conversion of hyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable in medicine. The gene that codifies for Hyoscyamine 6-β hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from <it>Brugmansia candida</it>.</p> <p>Results</p> <p>The <it>h6h</it>cDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in <it>Saccharomyces cerevisiae </it>in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the <it>h6h</it>cDNA.</p> <p>Conclusion</p> <p>The strains obtained in this work are promising and potentially applicable in biocatalytic processes.</p

Gorceixia decurrens (Compositae: Vernonieae): nova espécie para o estado da Bahia, Brasil

Recent fieldwork and collections have added a new genus to the Compositae flora of Bahia State, Brazil. Gorceixia decurrens is newly recorded for the State from Caatinga woodland along the lower part of the Estrada Real, in the municipality of Rio de Contas. A full description is provided, its distribution and conservation status discussed; likely affinities in the Vernonieae are discussed with the conclusion that it belongs to the subtribe Piptocarphinae.Trabalhos de campo e coletas recentes na Bahia, Brasil, permitiram adicionar mais um gênero para a flora do Estado. Gorceixia decurrens foi coletada em 2001, em área de Caatinga Arbórea, na base da Estrada Real no município a Rio de Contas. É fornecida uma completa descrição da espécie e discussão sobre sua distribuição e estado de conservação. Também, é apresentada discussão do posicionamento do gênero monotípico nas Vernonieae concluindo-se que o mesmo pertence à subtribo Piptocarphinae

Evidência de hibridação e introgressão em Hybanthus arenarius Ule e H. calceolaria (L.) Oken (Violaceae) a partir de marcadores aloenzimáticos e análise multivariada morfométrica

Allozyme and multivariate morphometric analyses using 46 morphological characters were carried out in 41 individuals of Hybanthus arenarius, 25 individuals of H. calceolaria and 33 morphologically intermediate individuals occurring on the campus of the Universidade Estadual de Feira de Santana to quantify the variability within these populations and to test hypotheses of hybridization and introgression. H. arenarius could be well differentiated in the morphometric analysis from both intermediate and H. calceolaria individuals, but the latter two groups could not be separated. Those same relationships were found in the allozyme analysis. The lack of diagnostic loci does not allow definitive confirmation of natural hybridization between the two species. However, several results found in the morphometric and genetic analyses reinforce the hypothesis that the individuals considered formerly as intermediate are actually individuals of H. calceolaria introgressed from H. arenarius.Foram realizadas análise morfométrica multivariada baseada em 46 caracteres morfológicos e análise de aloenzimas em 41 indivíduos de Hybanthus arenarius, 25 indivíduos de H. calceolaria e 33 indivíduos morfologicamente intermediários ocorrentes no campus da Universidade Estadual de Feira de Santana para determinar a variabilidade nestas populações e investigar hipóteses de hibridação e introgressão. Na análise morfométrica, H. arenarius pode ser bem diferenciada dos indivíduos intermediários e H. calceolaria, enquanto estes dois últimos não podem ser bem separados. Estas mesmas relações foram aparentes também na análise de aloenzimas. A ausência de loci diagnósticos não permite a confirmação de hibridação entre as espécies, mas vários resultados encontrados nas análises morfométrica e genética fortalecem a hipótese de que os indivíduos considerados primariamente como intermediários sejam indivíduos de H. calceolaria com introgressão de genes de H. arenarius

Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of Brugmansia candida (Solanaceae)

Brugmansia candida (Solanaceae) is a native tree distributed across South-American and produces the pharmacologically- important group of tropane alkaloids including scopolamine. This biocompound is synthesised from hyoscyamine by action of Hyoscyamine 6-\u3b2 hydroxylase (H6H, EC 1.14.11.11) at the end of the tropane alkaloid pathway. Here are reported the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the cDNA obtained from B. candida anthers and hairy root transformed cultures. Bioinformatic analysis of the nucleotide sequence revealed an uninterrupted ORF of 1038 bp and the predicted aminoacid sequence could be 344 aminoacid long. A database search showed that this sequence has high homology (97% identity) to Hyoscyamus niger H6H protein (Genbank accession number AAA33387.1)

Down-scale of hydrodynamic shear effects on suspended cells

Paper presented at the 9th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Malta, 16-18 July, 2012.The effect of shear forces on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, recent advances in the understanding of power consumption during agitation of Erlenmeyer flasks in rotary shakers are applied to direct comparison of the effects of shear forces on suspension cultures of Rubia tinctorum cells in a standard bioreactor and in shake flask cultures. The criterion selected as indicative of the shear stress acting on the cells was the critical eddy length, l, following Kolmogoroff's theory of isotropic turbulence. Standard flasks and four-baffled shake flasks were used. The effect of shear stress and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved previously in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm. It can be concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument to study intracellular mechanisms of shear stress-induced AQs production in R. tinctorum suspension cultures, including intracellular phenomena, and can be eventually extended to other systems.dc201

Filogenia molecular, morfologia e suas implicações para a taxonomia de Eriocaulaceae

The pantropical family Eriocaulaceae includes ten genera and c. 1,400 species, with diversity concentrated in the New World. The last complete revision of the family was published more than 100 years ago, and until recently the generic and infrageneric relationships were poorly resolved. However, a multi-disciplinary approach over the last 30 years, using morphological and anatomical characters, has been supplemented with additional data from palynology, chemistry, embryology, population genetics, cytology and, more recently, molecular phylogenetic studies. This led to a reassessment of phylogenetic relationships within the family. In this paper we present new data for the ITS and trnL-F regions, analysed separately and in combination, using maximum parsimony and Bayesian inference. The data confirm previous results, and show that many characters traditionally used for differentiating and circumscribing the genera within the family are homoplasious. A new generic key with characters from various sources and reflecting the current taxonomic changes is presented

Blind Testing: DNA Barcoding Sheds Light Upon the Identity of Plant Fragments as a Subsidy for Cave Conservation

Plants living above and around caves represent an important, albeit poorly studied, resource within cave ecosystems. The presence of plant material (root-like structures or rhizothemes, saplings, seeds, and seedlings) correlates positively with the biodiversity of the cave dwelling animals as shown for iron-ore caves in Carajás, Pará, Brazil. Plant material collected in caves has proven to be difficult to identify by traditional botanical methods, thus this research aims to provide a qualitative insight into the taxonomy and morphology of rhizothemes and other plant fragments found in the caves. The identification process used a combination of different molecular markers (ITS2, rbcL, and trnH-psbA) followed by a comparison of the sequences obtained against publicly available databases. The rhizothemes were submitted to micromorphological analysis to ascertain their putative root or stem origin and to compare their anatomy with known patterns found in the plant families or genera recovered through molecular matches. All studied samples were Angiosperms, mostly belonging to subclass Rosideae, within four orders: Malpighiales (Euphorbiaceae, Hypericaceae), Sapindales (Anacardiaceae and Sapindaceae), Myrtales (Myrtaceae), Fabales (Fabaceae), and only two belonging to subclass Asteridae, order Gentianales (Apocynaceae). Some of the samples were matched to generic level, with ITS2 being the best marker to identify the fragments because it shows high degree of sequence variation even at specific level and result reliability. All rhizothemes turned out to be roots, and correspondence was found between the existing literature and the individual anatomical patterns for the families and genera retrieved. DNA barcode has proved to be a useful tool to identify plant fragments found in this challenging environment. However, the existence of well curated, authoritatively named collections with ample biological information has proven to be essential to achieve a reliable identification