Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

<p>Abstract</p> <p>Background</p> <p>Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in <it>Nicotiana benthamiana </it>was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system.</p> <p>Results</p> <p>A <it>LOX </it>gene which is expressed after treatment of the viral vectors was isolated from <it>Nicotiana benthamiana</it>. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as <it>Nb-9-LOX</it>. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml^-1cell cultures) which can oxygenate linoleic acid resulting in high yields (18 μmol ml^-1cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C₉-aldehydes (3.3 μmol mg^-1crude protein). The yield of C₉-aldehydes from linoleic acid was 64%.</p> <p>Conclusion</p> <p>The yeast expressed Nb-9-LOX can be used to produce C₉-aldehydes on a large scale in combination with a <it>HPL </it>gene with 9-HPL function, or to effectively produce 9-hydroxy-10(<it>E</it>),12(<it>Z</it>)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent.</p

Atopic dermatitis : a cutaneous or systemic disease? The search for answers in the history of Dermatology

A dermatite atópica é doença inflamatória cutânea associada à atopia, predisposição a produzir resposta IgE a alérgenos ambientais, constituindo uma das manifestações das doenças atópicas, junto com a asma e a rinite alérgica. A dermatite atópica é caracterizada por episódios recorrentes de eczema associado a prurido, acometendo superfície cutânea geneticamente alterada, induzindo, por fenômenos imunológicos, a presença de inflamação. Trata-se de doença multifatorial, com enfoque nas alterações sistêmicas e alérgicas ou nas manifestações cutâneas, de acordo com diferentes visões da doença. A conceituação da dermatite atópica é importante, porque a conduta terapêutica pode variar segundo essas duas formas diferentes de analisá-la. Autores modernos discutem extensivamente esses aspectos sem, contudo, alcançar uma conclusão sobre a dermatite atópica como doença sistêmica ou cutânea. A procura dos conceitos sobre a doença, desde os primeiros relatos, associada à evolução do pensamento na dermatologia, poderia esclarecer a origem dessas dúvidas. Uma análise histórica demonstra que a dermatite atópica tem seus conceitos atuais oriundos dos estudos de diversos pensadores, que, em diferentes momentos históricos, descreveram a doença, e que muito do que acreditamos atualmente tem, nesses escritos, seus fundamentos.Atopic dermatitis is an inflammatory disease associated to atopy, which is a predisposition to produce an IgE response to environmental allergens and considered one of the manifestations of the atopic diseases, including asthma and allergic rhinitis. Atopic dermatitis is characterized by recurrent eczema flares, associated to pruritus, affecting a genetically disrupted skin surface, inducing, by immunological phenomena, the onset of inflammation. It is a multifactorial disease, with an emphasis on systemic and allergic alterations or skin manifestations, according to different concepts. The definition of atopic dermatitis is important, since its management may vary according to these two different points of view. Modern authors have extensively discussed these concepts, though with no conclusion as to its nature - systemic or cutaneous disease. The search for concepts about the disease, since its first descriptions, associated to the evolution of the dermatology rationale through history, may help understand the origin of these doubts. A historical analysis demonstrates that the currently accepted concepts of atopic dermatitis have their background from different researchers, who, at different historical moments, described the disease, and a great part of our beliefs about atopic dermatitis are related to these ancient writings