Desenvolvimento e validação de métodos bioanalíticos para o monitoramento dos produtos das vias metabólicas do triptofano em linhagem celular de glioma humano

O metabolismo do triptofano (Trp) se dá pela via das quinureninas (QUIN), pela via serotoninérgica (SER) e pela via das aminas traço. A primeira gera QUIN e uma variedade de outros metabólitos secundários. Quando conduzida pela enzima indolamina 2,3 dioxigenase (IDO) contribui para os fenômenos de tolerância e imune escape de células tumorais; e quando conduzida pela triptofano 2,3 dioxigenase (TDO) no fígado, participa na síntese da niacina e NAD. A via SER leva à formação do neurotransmissor serotonina (SER), que pode gerar o hormônio melatonina (MEL), respectivamente e outros metabólitos biologicamente ativos. Outra via menos estudada, a via das aminas traço, produz produtos neuroativos. Dada a abrangência e importância das rotas metabólicas do Trp, nós desenvolvemos e validamos uma metodologia bioanalítica robusta, seletiva e sensível por cromatografia líquida de alta eficiência (HPLC), acoplado espectrometria de massas (MS) para a determinação simultânea do Trp e seus 15 metabólitos. Para tanto, escolhemos para a avaliação das três vias, linhagens de glioma humano. A escolha por este tipo celular deveu-se ao grande interesse de estudos de metabolismo de Trp em células tumorais, no qual células de glioma tem sido modelo. Nos ensaios com as células de glioma acompanhamos os efeitos de um indutor e inibidores da primeira etapa de metabolização do Trp pela via das quinureninas, ou seja, IFN-γ (indutor da IDO), 1-metiltriptofano (1-MT; inibidor competitivo da IDO) e 680C91 (inibidor seletivo da TDO). Pudemos observar o impacto que a indução ou a inibição do primeiro passo teve sobre os metabólitos subsequentes e as diferenças no metabolismo das duas linhagens estudadas, A172 e T98G. A linhagem T98G só tem atividade de IDO, enquanto que a A172 tem tanto atividade IDO quanto TDO. A indução por IFN-γ mostrou que essa citocina não só atua na formação da via QUIN, mas possui um impacto modesto nas demais rotas. Observamos também que a inibição do 1-MT mostrou seu impacto nos metabólitos invdividualmente, do que a simples relação Trp-QUIN. Contudo, nosso resultados nos permitiu mostrar pela primeira vez a descrição completa dessas vias, em especial nessas linhagens celulares, podendo supor estratégias terapêuticas nessas rotas que estão relacionadas a progressão ou não tumoral.

Avaliação do potencial fotoprotetor e identificação de metabólitos secundários do fungo endofítico Annulohypoxylon stygium associado à alga marinha Bostrychia radicans

Os organismos marinhos constituem uma fonte potencial de metabólitos secundários biologicamente ativos. Neste contexto, os micro-organismos isolados de algas marinhas, dentre eles fungos endofíticos, representam alvos para a pesquisa de novas substâncias com potencial farmacológico pronunciado. Substâncias naturais provenientes de espécies de fungos associados às algas marinhas vêm sendo bastante utilizadas em formulações fotoprotetoras devido à ação antioxidante e ao potencial contra a radiação solar. Deste modo, o presente trabalho teve como objetivo a investigação biológica e química dos fungos endofíticos marinhos pertencentes à família Xylariaceae, o Annulohypoxylon stygium, o Cladosporium sp. e o Acremonium implicatum (Hypocreaceae). A princípio, foi realizado um screening para avaliar a absorção de luz ultravioleta na faixa do UVA e UVB pelos extratos obtidos em escala piloto destes fungos endofíticos associados às algas marinhas. O extrato do fungo A. stygium apresentou intensa absorção na região do UV, mostrando-se promissor para a produção de metabólitos secundários com ação fotoprotetora. Além do ensaio proposto, foi realizada a avaliação do potencial antibacteriano e antifúngico da espécie A. stygium. O estudo químico em escala ampliada deste fungo proporcionou o isolamento e identificação de uma substância inédita da classe derivada da 2,5- dicetopiperazina, 3-benzilideno-2-metil-hexahidro-pirrolo [1,2-?] pirazina-1,4-diona (Sf3), e além desta, foram isolados mais quatro metabólitos como, os diasteroisômeros 1-fenil-1,2- propanediol (Sd2) e 1-fenil-1,2-propanediol (Sd3), 1,3-benzodioxole-5-metanol (Sc1), 1,2- propanodiol-1-(1,3-benzodioxol-5-il) (Se1). Ainda foi possível a desreplicação de substâncias via cromatografia gasosa acoplada à espectrometria de massas (CG-EM), entre elas o ácido palmítico, palmitato de metila, ácido metil linoléico, ácido oléico, álcool benzílico e o piperonal. Quanto ao estudo da atividade biológica, não foi observado potencial antibacteriano e antifúngico para os extratos e frações do fungo. Entretanto, notouse um potencial como fotoprotetor in vitro para as frações n-Hexano/AcOEt (2:3) e n- Hexano/AcOEt (1:4) obtidas a partir do extrato do cultivo de 28 dias do fungo A. stygium, extraído com solventes diclorometano/metanol (CH2Cl2/MeOH 2:1) e para a substância (Sf3) isolada do mesmo. Desta forma, o estudo químico e biológico do fungo Annulohypoxylon stygium demonstrou potencial para a produção de metabólitos secundários com atividade fotoprotetora, visto que uma estrutura inédita com esta atividade foi isolada e identificada como produto natural.

Avaliação de parâmetros de qualidade em três variedades de mirtilo em modo de produção biológico e convencional

Nos últimos anos tem havido um aumento significativo da procura de frutos vermelhos. Os mirtilos são considerados frutos de boa qualidade, dado o seu elevado teor em compostos fitoquímicos biologicamente ativos, associados a efeitos benéficos para a saúde e bem-estar do Homem. A produção em modo biológico é reconhecida pelo consumidor como um processo que melhora a qualidade do produto. No presente trabalho pretendeu-se avaliar o efeito do modo de produção (biológico versus convencional) de três cultivares de mirtilo (Duke, Bluecrop, Ozarkblue) nas suas propriedades físico-químicas, e em particular na sua composição fenólica e atividade antioxidante. Foi ainda estudado o efeito da temperatura de armazenamento (± 5ºC e ± 15-25ºC) sobre essas propriedades. Para tal, as amostras foram analisadas à colheita e após 7 e 14 dias de armazenamento. A atividade antioxidante medida pelos métodos ABTS e DPPH mostrou que não há diferenças significativas entre as cultivares estudadas, sendo elevada em todos os casos. Os resultados obtidos confirmam, por isso, que o mirtilo é uma importante fonte de compostos fenólicos com elevada atividade antioxidante. Foi ainda verificado existirem algumas diferenças significativas em algumas propriedades em função da variedade (nomeadamente teor em matéria seca, cor ou textura). Também se verificara diferenças significativas em função do modo de produção, o qual influencia em particular a acidez e a doçura, o teor em taninos, a cor e a elasticidade dos frutos. Por fim, a temperatura de armazenamento mostrou ter uma influência significativa apenas no que respeita às propriedades físicas, nomeadamente cor e textura.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

Competitive sorption reactions between phosphorus and organic matter in soil: a review

The incorporation of organic matter ( OM) in soils that are able to rapidly sorb applied phosphorus ( P) fertiliser reportedly increases P availability to plants. This effect has commonly been ascribed to competition between the decomposition products of OM and P for soil sorption sites resulting in increased soil solution P concentrations. The evidence for competitive inhibition of P sorption by dissolved organic carbon compounds, derived from the breakdown of OM, includes studies on the competition between P and (i) low molecular weight organic acids (LOAs), (ii) humic and fulvic acids, and (iii) OM leachates in soils with a high P sorption capacity. These studies, however, have often used LOAs at 1 - 100 mM, concentrations much higher than those in soils ( generally < 0.05 mM). The transience of LOAs in biologically active soils further suggests that neither their concentration nor their persistence would have a practical benefit in increasing P phytoavailability. Higher molecular weight compounds such as humic and fulvic acids also competitively inhibit P sorption; however, little consideration has been given to the potential of these compounds to increase the amount of P sorbed through metal - chelate linkages. We suggest that the magnitude of the inhibition of P sorption by the decomposition products of OM leachate is negligible at rates equivalent to those of OM applied in the field. Incubation of OM in soil has also commonly been reported as reducing P sorption in soil. However, we consider that the reported decreases in P sorption ( as measured by P in the soil solution) are not related to competition from the decomposition products of OM breakdown, but are the result of P release from the OM that was not accounted for when calculating the reduction in P sorption.

The crystal structure of a bacterial Class II ketol-acid reductolsomerase: Domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

A review of the potential role of tumour-promoting compounds produced by Lyngbya majuscula in marine turtle fibropapillomatosis

Harmful algal blooms (HABs) have increased in abundance and severity in recent decades. Whereas the implications for human impacts and intoxication resulting from blooms have been extensively studied, the ecological implications of these microalgae are less well understood. Many HAB species produce biologically active, secondary metabolites and the fate of these toxins through the foodweb is generally not well understood unless it culminates in extensive fish mortalities or human poisonings. This review focusses on one HAB species, the cyanobacterium Lyngbya majuscula, and presents a hypothetical role for its involvement in fibro-papillornatosis (FP), a neoplastic disease of marine turtles. FP is expressed as benign tumours that grow both internally and externally on marine turtles, preventing vision, movement and organ function. The aetiology of FP is currently not conclusively understood, but virus material has been associated with tumours and previous studies have suggested a role for naturally produced tumour promoters. In this review, we present a hypothesis regarding the involvement of L. majuscula in FP, either through direct intoxication and action of tumour-promoting compounds or indirectly by causing seagrass loss and compromised immune function, thus leaving the turtles more susceptible to disease.

Complement factor 5a as a therapeutic target

The complement system is an innate immune defense mechanism that protects the host from infection and injury. Complement activation results in the formation of anaphylatoxins, including the biologically active protein C5a. This anaphylatoxin is a potent chemotactic agent for immune and inflammatory cells and induces cell activation. In situations of excessive or uncontrolled complement activation, the overproduction of C5a can cause deleterious effects to the host, and this process is implicated in the pathogenesis of numerous immunoinflammatory disease states, including rheumatoid arthritis, psoriasis, inflammatory bowel disease, ischemia-reperfusion injuries and others. The presence of C5a in a wide variety of condition's has prompted many groups to examine the potential of inhibiting this complement activation product, with the aim of controlling these diseases and reducing the pathologic process. However, to date there is no clinically available specific C5a inhibitor and development of this new drug class is still in a relatively early stage, although limited phase I and phase II human clinical trials have been undertaken in the last few years with selected agents. In this review, examination of the current evidence supporting a specific role of C5a in selected disease states and an overview of potential therapeutic C5a inhibitors will enable the critical evaluation of the potential for C5a as a therapeutic target.

Criteria for quantum coherent transfer of excitations between chromophores in a polar solvent

We show that the quantum decoherence of Forster resonant energy transfer between two optically active molecules can be described by a spin-boson model. This allows us to give quantitative criteria that are necessary for coherent quantum oscillations of excitations between the chromophores. Experimental tests of our results should be possible with flourescent resonant energy transfer (FRET) spectroscopy. Although we focus on the case of protein-pigment complexes our results are also relevant to quantum dots and organic molecules in a dielectric medium. (c) 2006 Elsevier B.V. All rights reserved.

Kidney dysfunction and hypertension: Role for cadmium, P450 and heme oxygenases?

Cadmium (Cd) is a metal toxin of continuing worldwide concern. Daily intake of Cd, albeit in small quantities, is associated with a number of adverse health effects which are attributable to distinct pathological changes in a variety of tissues and organs. In the present review, we focus on its renal tubular effects in people who have been exposed environmentally to Cd at levels below the provisional tolerable intake level set for the toxin. We highlight the data linking such low-level Cd intake with tubular injury, altered abundance of cytochromes P450 (CYPs) in the kidney and an expression of a hypertensive phenotype. We provide updated knowledge on renal and vascular effects of the eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and eicosatrienoic acids (EETs), which are biologically active metabolites from arachidonate metabolism mediated by certain CYPs in the kidney. We note the ability of Cd to elicit oxidative stress and to alter metal homeostasis notably of zinc which may lead to augmentation of the defense mechanisms involving induction of the antioxidant enzyme heme oxygenase-1 (HO-1) and the metal binding protein metallothionein (MT) in the kidney. We hypothesize that renal Cd accumulation triggers the host responses mediated by HO-I and MT in an attempt to protect the kidney against injurious oxidative stress and to resist a rise in blood pressure levels. This hypothesis predicts that individuals with less active HO-1 (caused by the HO-1 genetic polymorphisms) are more likely to have renal injury and express a hypertensive phenotype following chronic ingestion of low-level Cd, compared with those having more active HO-1. Future analytical and molecular epidemiologic research should pave the way to the utility of induction of heme oxygenases together with dietary antioxidants in reducing the risk of kidney injury and hypertension in susceptible people.

Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays. (C) 2005 Elsevier SAS. All rights reserved.

Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design

The cyclotide family of plant proteins is of interest because of their unique topology, which combines a head-to-tail cyclic backbone with an embedded cystine knot, and because their-remarkable chemical and biological properties make them ideal candidates as grafting templates for biologically active peptide epitopes. The present Study describes the first steps towards exploiting the cyclotide framework by synthesizing and structurally characterizing two grafted analogues of the cyclotide kalata B1. The modified peptides have polar or charged residues substituted for residues that form part of a surface-exposed hydrophobic patch that plays a significant role in the folding and biological activity of kalata B1. Both analogues retain the native cyclotide fold, but lack the undesired haemolytic activity of their parent molecule, kalata B1. This finding confirms the tolerance of the cyclotide framework to residue Substitutions and opens up possibilities for the Substitution of biologically active peptide epitopes into the framework.

Inorganic nanoparticles as carriers for efficient cellular delivery

Cellular delivery involving the transfer of various drugs and bio-active molecules (peptides, proteins and DNAs, etc.) through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. This topic has been extensively reviewed. The direct delivery of drugs and biomolecules, however, is generally inefficient and suffering from problems such as enzymic degradation of DNAs. Therefore, searching for efficient and safe transport vehicles (carriers) to delivery genes or drugs into cells has been challenging yet exciting area of research. In past decades, many carriers have been developed and investigated extensively which can be generally classified into four major groups: viral carriers, organic cationic compounds, recombinant protiens and inorganic nanoparticles. Many inorganic materials, such as calcium phosphate, gold, carbon materials, silicon oxide, iron oxide and layered double hydroxide (LDH), have been studied. Inorganic nanoparticles show low toxicity and promise for controlled delivery properties, thus presenting a new alternative to viral carriers and cationic carriers. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal tissues) and controlled release of carried drugs. This paper reviews the latest advances in inorganic nanoparticle applications as cellular delivery carriers and highlights some key issues in efficient cellular delivery using inorganic nanoparticles. Critical proper-ties of inorganic nanoparticles, surface functionalisation (modification), uptake of biomolecules, the driving forces for delivery, and release of biomolecules will be reviewed systematically. Selected examples of promising inorganic nanoparticle delivery systems, including gold, fullerences and carbon nanotubes, LDH and various oxide nanoparticles in particular their applications for gene delivery will be discussed. The fundamental understanding of properties of inorganic nanoparticles in relation to cellular delivery efficiency as the most paramount issue will be highlighted. (c) 2005 Elsevier Ltd. All rights reserved.

S. cerevisiae as a model for studying mutations in the human gene OPA1 associated with dominant optic atrophy and for drug discovery

L’atrofia ottica dominante (ADOA) è una malattia mitocondriale caratterizzata da difetti visivi, che si manifestano durante l’infanzia, causati da progressiva degenerazione delle cellule gangliari della retina (RGC). ADOA è una malattia genetica associata, nella maggior parte dei casi, a mutazioni nel gene OPA1 che codifica per la GTPasi mitocondriale OPA1, appartenente alla famiglia delle dinamine, principalmente coinvolta nel processo di fusione mitocondriale e nel mantenimento del mtDNA. Finora sono state identificate più di 300 mutazioni patologiche nel gene OPA1. Circa il 50% di queste sono mutazioni missenso, localizzate nel dominio GTPasico, che si pensa agiscano come dominanti negative. Questa classe di mutazioni è associata ad una sindrome più grave nota come “ADOA-plus”. Nel lievito Saccharomyces cerevisiae MGM1 è l’ortologo del gene OPA1: nonostante i due geni abbiano domini funzionali identici le sequenze amminoacidiche sono scarsamente conservate. Questo costituisce una limitazione all’uso del lievito per lo studio e la validazione di mutazioni patologiche nel gene OPA1, infatti solo poche sostituzioni possono essere introdotte e studiate nelle corrispettive posizioni del gene di lievito. Per superare questo ostacolo è stato pertanto costruito un nuovo modello di S. cerevisiae, contenente il gene chimerico MGM1/OPA1, in grado di complementare i difetti OXPHOS del mutante mgm1Δ. Questo gene di fusione contiene una larga parte di sequenza corrispondente al gene OPA1, nella quale è stato inserito un set di nuove mutazioni trovate in pazienti affetti da ADOA e ADOA-plus. La patogenicità di queste mutazioni è stata validata sia caratterizzando i difetti fenotipici associati agli alleli mutati, sia la loro dominanza/recessività nel modello di lievito. A tutt’oggi non è stato identificato alcun trattamento farmacologico per la cura di ADOA e ADOA-plus. Per questa ragione abbiamo utilizzato il nostro modello di lievito per la ricerca di molecole che agiscono come soppressori chimici, ossia composti in grado di ripristinare i difetti fenotipici indotti da mutazioni nel gene OPA1. Attraverso uno screening fenotipico high throughput sono state testate due differenti librerie di composti chimici. Questo approccio, noto con il nome di drug discovery, ha permesso l’identificazione di 23 potenziali molecole attive.

Increased TG2 expression can result in induction of transforming growth factor β1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.