RESUMO
Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.
Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Chaperonina 60/administração & dosagem , Chaperonina 60/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Mycobacterium leprae/enzimologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Feminino , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lactococcus lactis/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don't need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology.
Assuntos
Biocombustíveis , Lipídeos/biossíntese , Engenharia Metabólica/métodos , Microalgas , Fermentação , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismoRESUMO
Malaria is a devastating disease depending only on chemotherapy as treatment. However, medication is losing efficacy, and therefore, there is an urgent need for the discovery of novel pharmaceutics. Recently, plasmepsin V, an aspartic protease anchored in the endoplasmaic reticulum, was demonstrated as responsible for the trafficking of parasite-derived proteins to the erythrocytic surface and further validated as a drug target. In this sense, ligand-based virtual screening has been applied to design inhibitors that target plasmepsin V of P. falciparum (PMV). After screening 5.5 million compounds, four novel plasmepsin inhibitors have been identified which were subsequently analyzed for the potency at the cellular level. Since PMV is membrane-anchored, the verification in vivo by using transgenic PMV overexpressing P. falciparum cells has been performed in order to evaluate drug efficacy. Two lead compounds, revealing IC50 values were 44.2 and 19.1 µm, have been identified targeting plasmepsin V in vivo and do not significantly affect the cell viability of human cells up to 300 µm. We herein report the use of the consensus of individual virtual screening as a new technique to design new ligands, and we propose two new lead compounds as novel protease inhibitors to target malaria.
Assuntos
Antimaláricos/química , Ácido Aspártico Endopeptidases/metabolismo , Ligantes , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Organismos Geneticamente Modificados/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genéticaRESUMO
To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.
Assuntos
Acinetobacter/metabolismo , Biodegradação Ambiental , Citocromo P-450 CYP4A/genética , Escherichia coli/metabolismo , Consórcios Microbianos/genética , Organismos Geneticamente Modificados/metabolismo , Pseudomonas putida/enzimologia , Acinetobacter/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Óleos Combustíveis , Gasolina , Engenharia Genética , Organismos Geneticamente Modificados/genética , Oxirredução , Plasmídeos/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMO
To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (
Assuntos
Acinetobacter/metabolismo , Biodegradação Ambiental , /genética , Escherichia coli/metabolismo , Consórcios Microbianos/genética , Organismos Geneticamente Modificados/metabolismo , Pseudomonas putida/enzimologia , Acinetobacter/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Óleos Combustíveis , Gasolina , Engenharia Genética , Oxirredução , Organismos Geneticamente Modificados/genética , Plasmídeos/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMO
Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.
Assuntos
Fosfatase Ácida/metabolismo , Metabolismo dos Carboidratos , Enterobacteriaceae/enzimologia , Nucleosídeos/metabolismo , Fosfotransferases/metabolismo , Enterobacteriaceae/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Fatores de TempoRESUMO
Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.
Assuntos
Proteínas de Fluorescência Verde/biossíntese , Trypanosoma cruzi/fisiologia , Animais , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/genética , Interações Hospedeiro-Parasita , Concentração Inibidora 50 , Viabilidade Microbiana , Nitroimidazóis/farmacologia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Organismos Geneticamente Modificados/fisiologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
This article gives an overview of high-cell-density cultures for polyhydroxyalkanoate (PHA) production and their modes of operation for increasing productivity. High cell densities are very important in PHA production mainly because this polymer is an intracellular product accumulated in various microorganisms, so a high cellular content is needed for the polymer production. This review describes relevant results from fed-batch, repeated batch, and continuous modes of operation without and with cell recycle for the production of these polymers by microorganisms. Finally, recombinant microorganisms for PHA production, as well future directions for PHA production, are discussed.
Assuntos
Técnicas de Cultura Celular por Lotes , Poli-Hidroxialcanoatos/biossíntese , Organismos Geneticamente Modificados/metabolismoRESUMO
Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC(50) values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Leishmania/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Testes de Sensibilidade Parasitária/métodos , Anfotericina B/farmacologia , Animais , Antiprotozoários/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração Inibidora 50 , Leishmania/genética , Leishmania/metabolismo , Proteínas Luminescentes/genética , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Propranolol/análogos & derivados , Propranolol/farmacologia , Reprodutibilidade dos Testes , Transfecção , Proteína Vermelha FluorescenteRESUMO
Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level, and its absence is involved in inflammatory bowel diseases. However, oral treatment with IL-10 is difficult because of its low survival in the gastrointestinal tract and systemic treatments lead to undesirable side effects. The aim of this paper was to evaluate the anti-inflammatory effect of the administration of milks fermented by Lactococcus lactis strains that produce IL-10 under the control of the xylose-inducible expression system using a trinitrobenzenesulfonic acid-induced colitis murine model. Mice that received milks fermented by L. lactis strains producing IL-10 in the cytoplasm (Cyt strain) or secreted to the product (Sec strain) showed lower damage scores in their large intestines, decreased IFN-γ levels in their intestinal fluids and lower microbial translocation to liver, compared to mice receiving milk fermented by the wild-type strain or those not receiving any treatment. The results obtained in this study show that the employment of fermented milks as a new form of administration of IL-10-producing L. lactisis effective in the prevention of inflammatory bowel disease in a murine model.
Assuntos
Anti-Inflamatórios/análise , Doença de Crohn/terapia , Dieta/métodos , Interleucina-10/análise , Lactococcus lactis/metabolismo , Leite/química , Animais , Translocação Bacteriana , Colite/induzido quimicamente , Colite/patologia , Colite/terapia , Doença de Crohn/patologia , Modelos Animais de Doenças , Fezes/química , Fermentação , Interferon gama/análise , Lactococcus lactis/genética , Fígado/microbiologia , Camundongos , Leite/microbiologia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Proteínas Recombinantes/análise , Índice de Gravidade de DoençaRESUMO
The phytostimulatory properties of Azospirillum inoculants, which entail production of the phytohormone indole-3-acetic acid (IAA), can be enhanced by genetic means. However, it is not known whether this could affect their interactions with indigenous soil microbes. Here, wheat seeds were inoculated with the wild-type strain Azospirillum brasilense Sp245 or one of three genetically modified (GM) derivatives and grown for one month. The GM derivatives contained a plasmid vector harboring the indole-3-pyruvate/phenylpyruvate decarboxylase gene ipdC (IAA production) controlled either by the constitutive promoter PnptII or the root exudate-responsive promoter PsbpA, or by an empty vector (GM control). All inoculants displayed equal rhizosphere population densities. Only inoculation with either ipdC construct increased shoot biomass compared with the non-inoculated control. At one month after inoculation, automated ribosomal intergenic spacer analysis (ARISA) revealed that the effect of the PsbpA construct on bacterial community structure differed from that of the GM control, which was confirmed by 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). The fungal community was sensitive to inoculation with the PsbpA construct and especially the GM control, based on ARISA data. Overall, fungal and bacterial communities displayed distinct responses to inoculation of GM A. brasilense phytostimulators, whose effects could differ from those of the wild-type.
Assuntos
Azospirillum brasilense/metabolismo , Biodiversidade , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/microbiologia , Microbiologia do Solo , Triticum/microbiologia , Azospirillum brasilense/genética , Azospirillum brasilense/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Biomassa , Vias Biossintéticas/genética , Carboxiliases/genética , Impressões Digitais de DNA/métodos , DNA Espaçador Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Dosagem de Genes , Engenharia Genética , Metagenoma , Desnaturação de Ácido Nucleico , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/crescimento & desenvolvimento , Organismos Geneticamente Modificados/metabolismo , Brotos de Planta/crescimento & desenvolvimento , PlasmídeosRESUMO
Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited (14)C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice.