Cloning and Function Analysis of ω-3-Fatty Acid Desaturase 8 Gene from Perilla frutescens / 中国生物化学与分子生物学报

ω-3-Fatty acid desaturase 8 (FAD8), as a dehydrogenase enzyme, plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids, which is helpful to enhance the freezing tolerance of plants. However, it remains unclear whether the expression level of FAD8 in Perilla frutescens is regulated by low temperature. Based on transcriptome data, the FAD8 gene was cloned, characterized and then successfully expressed in tobacco Nicotiana tabacum. The gene was designated as PfFAD8 and has a full-length coding sequence of 1 317 bp coding for 438 amino acids with a predicted molecular weight of 50 kD and a theoretical isoelectric point of 9. 13. Our research indicated that the expression of PfFAD8 in Perilla frutescens was increased under the freezing stress. To further confirm this result, a 35S::PfFAD8 vector were constructed and transformed into N. tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco leaves that over-expressed the PfFAD8 gene exhibited significantly higher unsaturated fatty acids (UFA) such as linoleic (C18:2) and palmitic acid (C16:0) content and advanced freezing tolerance. Moreover, PfFAD8 overexpression in transgenic tobacco leaves increases malondialdehyde (MDA) and proline (PRO) content, and enhances defense enzymes activities of superoxide dismutase (SOD) and catalase (CAT) to some extent under the cold condition, which might prevent the decline of UFA. Taken together, PfFAD8 overexpression in Perilla frutescens might be involved in the desaturation process of lipids leading to increased membrane stability and/ or induction of other genes related to freezing tolerance by octadecanoid pathway or lipid peroxidation products. Thus, PfFAD8 overexpression could be useful in the production of freeze-tolerant varieties of N. tabacum.

Ciclos de atenção a dietas da moda e tendências de busca na internet pelo Google trends / Cycles of attention to fad diets and internet search trends by Google trends

Resumo Este trabalho objetivou descrever e analisar conteúdos de notícias associadas a ciclos de buscas ao Google ligados a 8 tipos de dieta: a cetogênica; da lua; da proteína; da sopa; detox; dos pontos; paleo; e Dukan. Estas foram selecionadas por terem sido apontadas pelo Google Trends® (GT) como as mais frequentemente associadas ao termo dieta. Os conteúdos dos sites vinculados aos maiores picos de buscas foram apontados pelo Google News®. O volume de buscas às dietas foram estimados pelo GT com filtros de região (Brasil) e temática (Saúde) considerando o período de 01/01/2012 a 01/01/2017. Os acessos retratam ciclos efêmeros com centenas de picos e quedas de interesse pelas 8 dietas da moda. A ênfase se concentra nas orientações para rápida perda de peso em prol de um ideal de saúde tido como proxy de um ideal estético. A dimensão dos riscos associados às dietas mais restritivas é citada frequentemente e validada por informações de especialistas. Destaca-se a exposição e a influência da opinião de celebridades relatando suas experiências dietéticas. Ao contrário do ideal de variedade e equilíbrio, as dietas da moda retratam a pressão pelo autocontrole alimentar como recurso à construção de um corpo idealizado e tipificado por imagens das celebridades.

Abstract This work aimed at identifying, describing and analyzing news content associated with Google search cycles linked to eight types of diet: ketogenic; moon; protein; soup; detox; points; paleo; and Dukan. They were selected because they were pointed out by Google Trends® (GT) as the most often associated with the term "diet". The content of sites linked to the highest search peaks was pointed out by Google News®. The volume of search attempts for diets was estimated by the GT with region filters (Brazil) and theme (Health) considering the period from 01/01/2012 to 01/01/2017. Results: hits portray ephemeral cycles, with hundreds of peaks and declining interest in the eight fad diets. The emphasis is on guidelines for quick weight loss in favor of optimal health as a proxy for an esthetic ideal. The scale of the risks associated with the more restrictive diets is often quoted and validated by expert information. The exposure and influence of the opinion of celebrities reporting their dietary experiences stand out. Contrary to the ideal of moderation, variety, and balance, fad diets portray the pressure for food self-control as a resource for building an idealized body typified by celebrity images.

Nutritional composition of fad diets published on websites and blogs / Composição nutricional de dietas da moda publicadas em sites e blogs

ABSTRACT Objective To analyze quantitatively the diets available on blogs and websites. Methods The following 15 diets were analyzed: low-carb diet, gluten-free diet and intermittent fasting diet using the Virtual Nutri software program. The nutrients analyzed were as follows: carbohydrates, fibers, proteins, lipids, iodine, sodium, calcium, magnesium, iron, zinc, manganese, phosphorus, selenium, vitamins A, C, B6, B12, D, niacin, and folate. The nutrient values were compared with the Dietary Reference Intake recommendation for adults between the ages of 19-50 years. The macronutrient adequacy percentage was based on a 2000-kcal diet for a healthy life, as recommended by the World Health Organization. Results It was observed that most of the composition of macro and micronutrients was lower than the one established by the World Health Organization and the Dietary Reference Intake recommendation for adults between the ages of 19-50 years. Conclusion Long-term dieting can be a risk factor for several deficiency diseases and may endanger the health of individuals.

RESUMO Objetivo Analisar quantitativamente dietas disponíveis em blogs e sites. Métodos Analisou-se 15 cardápios de dietas intituladas: Low Carb, Dieta Sem Glúten e Jejum Intermitente utilizando o software, Virtual Nutri. Os nutrientes analisados foram: carboidratos, fibras, proteínas, lipídios, iodo, sódio, cálcio, magnésio, ferro, zinco, manganês, potássio, fósforo, cobre, selênio, vitaminas A, C, B6, B12, D, niacina e folato. Os nutrientes tiveram seus valores comparados com recomendações diárias de ingestão das Dietary Reference Intake para adultos da faixa etária de 19 a 50 anos. O percentual de adequação foi feito baseado em uma dieta de 2000kcal para indivíduos saudáveis, de acordo com o preconizado pela Organização Mundial da Saúde. Resultados Observou-se que a maioria dos cardápios teve sua composição de macro e micronutrientes aquém do determinado pela Organização Mundial da Saúde e das recomendações das Dietary Reference Intake para adultos de 19 a 50 anos. Conclusão O seguimento dessas dietas em longo prazo pode ser um fator de risco a diversas doenças carenciais que pode trazer perigos à saúde dos indivíduos.

Recent trends in two-photon auto-fluorescence lifetime imaging (2P-FLIM) and its biomedical applications

Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing.

Expression analysis of genes involved in the synthesis of oleic and linoleic acids in Jatropha cinerea seeds from Northwestern Mexico / Análise de expressão de genes envolvidos na síntese de ácidos oleico e linoleico em sementes de Jatropha cinerea do noroeste do México

ABSTRACT: Plants belonging to genus Jatropha has arisen interest because of its high oil content that could be used to produce biodiesel. It is also widely reported that the main fatty acids in Jatropha oilseed are oleic and linoleic acids. However, there are scarce studies related to native species of Jatropha from Northwestern Mexico which are adapted to arid conditions, and the expression of genes involved in fatty acid synthesis for these species is still unknown. Therefore, the aim of this study was to analyze the expression of five genes, ACP1, KASII, D9SD, FAD2-1 and FAD2-2, which are involved in the oleic and linoleic acids synthesis in mature wild-seeds of Jatropha cinerea, a native species from Sonoran Desert, using semi-quantitative RT-PCR. The J. cinerea seeds were randomly collected in Bahía de Kino, Sonora (México) which is a region characterized by its harsh environments such as saline soils and extreme temperature changes and J. curcas mature seeds from a non-toxic variety from Veracruz, Mexico were used as a reference. The RT-PCR analysis of three biological replicates were considered to ensure data consistent. Our analysis showed a higher expression of KASII and FAD2-1 genes in J. cinerea seeds compared to J. curcas, meanwhile the expression of ACP1, D9SD and FAD2-2 were higher in J. curcas. Furthermore, Actin and FAD2-1 genes sequences here obtained are the first reported for J. cinerea, thus providing information to develop further studies.

RESUMO: Plantas pertencentes ao gênero Jatropha têm despertado interesse devido ao seu alto teor de óleo que poderia ser usado para produzir biodiesel. Também é amplamente relatado que os principais ácidos graxos das oleaginosas da Jatropha são os ácidos oléico e linoleico. No entanto, existem poucos estudos relacionados a espécies nativas de pinhão-manso do noroeste do México que estão adaptados a condições áridas, e a expressão de genes envolvidos na síntese de ácidos graxos para essas espécies ainda é desconhecida. Portanto, o objetivo deste estudo foi analisar a expressão de cinco genes, ACP1, KASII, D9SD, FAD2-1 e FAD2-2, que estão envolvidos na síntese de ácidos oleico e linoleico em sementes selvagens maduras de Jatropha cinerea, espécies nativas do Deserto de Sonora, usando RT-PCR semi-quantitativa. Sementes de J. cinerea foram coletadas aleatoriamente em Bahía de Kino, Sonora (México), região caracterizada por ambientes agressivos como solos salinos e mudanças extremas de temperatura, e sementes de J. curcas maduras de uma variedade não-tóxica de Veracruz, México, usado como referência. Análise RT-PCR de três repetições biológicas foram consideradas para garantir dados consistentes. Nossa análise mostrou uma maior expressão dos genes KASII e FAD2-1 em sementes de J. cinerea comparado a J. curcas, enquanto a expressão de ACP1, D9SD e FAD2-2 foi maior em J. curcas. Além disso, as sequências dos genes Actin e FAD2-1 obtidas são as primeiras relatadas para J. cinerea, fornecendo, assim, informações para o desenvolvimento de novos estudos.

Analysis of the peanut transgenic offspring with depressing AhFAD2 gene / 生物工程学报

The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.

Sodium azide mutagenesis resulted in a peanut plant with elevated oleate content

Screening of peanut seeds resulting from 0.39 percent sodium azide treatment with NIRS calibration equation for bulk seed samples identified a plant with more than 60 percent oleate. Oleate content in individual seeds of the plant, as predicted by NIRS calibration equation for intact single peanut seeds, ranged from 50.05 percent ~ 68.69 percent. Three seeds with >60 percent oleate thus identified were further confirmed by gas chromatography. Multiple sequence alignments of the FAD2B gene from Huayu 22 (wild type) and peanut seeds with elevated oleate (mutant type) revealed a C281T transition in the coding region causing an I94T substitution in the oleoyl-PC desaturase, which may be responsible for reduction in the enzyme activity.

Advances in Application Studies of FAD2 on Biosynthesis of Industrial Fatty Acids / 中国生物工程杂志

Many unusual fatty acids present in the seed oils of non-food plants are modified at the ?12 position either by the addition of epoxy or hydroxy groups or by the formation of triple (acetylenic) bonds or conjugated double bonds. The introduction of these functionalities into C18 fatty acids is catalyzed by a family of divergent forms of the fatty-acid ?12-desaturase (FAD2) enzyme. Genes encoding these enzymes have now been cloned from a variety of organisms and expressed in transgenic oilseeds. However,the accumulation of the intended fatty acids in transgenic plants has been relatively disappointing,and the mechanisms of these unusual fatty acids biosynthesis and transferring from phosphatidylcholine into storage triacylglycerols still need to be elucidated.

Expression and characterization of the flavoprotein domain of gp91phox

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).

Activation domain in P67phox regulates the steady state reduction of FAD in gp91phox

An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.

Naphthalene oxygenase from Corynebacterium renale : Characterisation and mechanism of oxygenation.

The formation of cis-l,2-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified from Corynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption ofone mol oxygen. The enzyme contains one g-atom Fe2+ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H2O2 was present; the generation of superoxide anion on addition ofNADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H2O2 was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction.

DETERMINATION OF RIBOFLAVIN,FMN,AND FAD IN HUMAN PLASMA AND ERYTHROCYTES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY / 营养学报

Objective: To develop a high performance liquid chromatographic (HPLC) method for determination of riboflavin, FMN, and FAD in human plasma and erythrocytes. Method: Waters 600 model HPLC pump and an Atlantis TM C18 column (150 mm?4.6 mm i.d. 5 ?m) were used. The mobile phase consisted of 35% methanol and 65% 5 mmol/L ammonium acetate solution. The flow rate was 1.0ml/min. The spectro-photofluorimeter was set at wavelength of 450 nm for excitation and 520 nm for emission. Plasma and erythrocyte hemolyzed samples were treated with acetonitrile and chloroform. and the supernatant was analyzed. Results: A good linear correlation existed between riboflavin, FMN and FAD concentration (from 1 to 400 nmol/L) and fluorescence intensity. The detection limits were 2.0 nmol/L, 2.5nmol/L and 2.5nmol/L at a signal to noise ratio of 3, respectively. The intra-assay and inter-assay relative standard deviations were in the range of 1.3% to 3.7%. The recoveries for riboflavin, FMN and FAD in both plasma and erythrocytes were satisfactory. Conclusion:This method for determination of riboflavin,FMN, and FAD in human plasma and erythrocytes is sensitive, rapid and accurate.