RESUMO
To validate therapeutic targets in metabolic pathways of trypanosomatids, the criterion of enzyme essentiality determined by gene knockout or knockdown is usually being applied. Since, it is often found that most of the enzymes/proteins analyzed are essential, additional criteria have to be implemented for drug target prioritization. Metabolic control analysis (MCA), often in conjunction with kinetic pathway modeling, offers such possibility for prioritization. MCA is a theoretical and experimental approach to analyze how metabolic pathways are controlled. It involves strategies to perform quantitative analyses to determine the degree in which an enzyme controls a pathway flux, a value called flux control coefficient ([Formula: see text]). By determining the [Formula: see text] of individual steps in a metabolic pathway, the distribution of control of the pathway is established, that is, the identification of the main flux-controlling steps. Therefore, MCA can help in ranking pathway enzymes as drug targets from a metabolic perspective. In this chapter, three approaches to determine [Formula: see text] are reviewed: (1) In vitro pathway reconstitution, (2) manipulation of enzyme activities within parasites, and (3) in silico kinetic modeling of the metabolic pathway. To perform these methods, accurate experimental data of enzyme activities, metabolite concentrations and pathway fluxes are necessary. The methodology is illustrated with the example of trypanothione metabolism of Trypanosoma cruzi and protocols to determine such experimental data for this metabolic process are also described. However, the MCA strategy can be applied to any metabolic pathway in the parasite and general directions to perform it are provided in this chapter.
Assuntos
Desenvolvimento de Medicamentos/métodos , Metabolômica/métodos , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Extratos Celulares/isolamento & purificação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Simulação por Computador , Glutationa/análogos & derivados , Glutationa/metabolismo , Humanos , Cinética , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espermidina/análogos & derivados , Espermidina/metabolismo , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacosRESUMO
BACKGROUND: Fitzroya cupressoides, commonly known as alerce, is an endemic conifer unique to southern South America. Alerce wood is renowned for its durability and resistance to biological degradation due to the presence of a particular class of secondary metabolite. Alerce extracts have been used in traditional medicine for different skin lesion treatments. AIMS: To develop a cell culture system to produce alerce extract and evaluate its cytotoxicity and effects on in vitro wound healing. METHODS: Cell cultures and aqueous extracts were prepared from alerce needles. Cytotoxicity was evaluated in keratinocytes (HaCaT line) and melanocites (C32 line) using the XTT assay. Wound healing was assayed with the scratch test in HaCaT cells, using mitomycin C to evaluate the role of cell division in the wound closure. RESULTS: Alerce cell culture extract has a significant effect on wound healing at different concentrations. No positive effects on the viability of normal and cancerous skin cells were observed. These results suggest that alerce extracts stimulate cell division in human skin epidermal cells in the context of wound repair. CONCLUSIONS: Bioactive compounds extracted from alerce cell cultures show promise as ingredients in dermocosmetic formulations, but further clinical studies are required to support these findings at the tissue level.
Assuntos
Extratos Celulares/farmacologia , Cosmecêuticos/farmacologia , Cupressaceae/química , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Técnicas de Cultura de Células , Extratos Celulares/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cosmecêuticos/isolamento & purificação , Cupressaceae/citologia , Humanos , Queratinócitos , Melanocortinas , Extratos Vegetais/isolamento & purificação , Testes de Toxicidade AgudaRESUMO
Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a-better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.
Assuntos
Movimento Celular , Extensões da Superfície Celular/fisiologia , Extensões da Superfície Celular/ultraestrutura , Entamoeba histolytica/fisiologia , Entamoeba histolytica/ultraestrutura , Trofozoítos/fisiologia , Trofozoítos/ultraestrutura , Extratos Celulares/isolamento & purificação , Extensões da Superfície Celular/efeitos dos fármacos , Entamoeba histolytica/efeitos dos fármacos , Eritrócitos/química , Fibronectinas/isolamento & purificação , Fibronectinas/metabolismo , Humanos , Microscopia , Microscopia Confocal , Microscopia Eletrônica de Varredura , Trofozoítos/efeitos dos fármacosRESUMO
Antimicrobial activities have previously been described by traditional Eastern medicine in Chilopoda body extracts, but until now no bioactive peptides have been described. In this study, a novel antimicrobial peptide, lacrain, was isolated from the body extract of the Brazilian Chilopoda Scolopendra viridicornis. The peptide was isolated by reverse-phase high-performance liquid chromatography (RP-HPLC). Its activity was tested using a liquid growth inhibition assay and the peptide was characterised using mass spectrometry. Lacrain has a sequence composed of eight amino acid residues and a molecular mass of 925.5 Da. A synthetic peptide of the native lacrain had identical characteristics to those of the isolated material, confirming its sequence. The synthetic peptide was active only against Gram-negative bacteria, showing strong bactericidal activity. Moreover, the peptide did not present haemolytic activity against human erythrocytes. Lacrain represents a novel molecule with powerful antibacterial activity that could be used as a new template for the development of drugs against clinically resistant bacterial strains.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Artrópodes/química , Extratos Celulares/farmacologia , Adulto , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Brasil , Extratos Celulares/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Eritrócitos/efeitos dos fármacos , Fungos/efeitos dos fármacos , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Peso Molecular , Análise de Sequência de ProteínaRESUMO
The royal sun mushroom, Agaricus brasiliensis is a widely consumed mushroom around the world. In this study, the immunoregulatory potential of A. brasiliensis polysaccharides was investigated in vitro and in vivo. In vivo, the polysaccharides remarkably increased the spleen and thymus indexes in mice, and this effect was influenced significantly by age (the adult and the juvenile). The spleen index increased by 27.28% in adult mice treated with the polysaccharides, whereas the increase in juvenile mice was just 12.59% at the dose of 150 mg·kg-1·d-1. Moreover, the effect of the polysaccharides on the thymus and spleen indexes in adult mice was obvious both in males and females. The carbon clearance ability (phagocytic index) was improved with increasing doses, (32.81% at 120 mg·kg-1·d-1, and 38.34% at 150 mg·kg-1·d-1) in mice treated with the polysaccharides. In vitro, the polysaccharides increased the RAW264.7 cell proliferation with 34.78% at 25 µg/mL and 26.78% at 50 µg/mL. Furthermore, the polysaccharides also promoted mRNA expressions of interleukin (IL)-6, IL-1ß, cyclooxygenase-2, and Toll-like receptor 4 (TLR4), myeloid differentiation 88 (MYD88), and TIR-domain-containing adapter-inducing interferon-ß (TRIF) in the cells, indicating that the polysaccharides induce the secretion of inflammatory cytokines by stimulating TLR4/MyD88 and TLR4/TRIF pathways. In conclusion, these results suggest that A. brasiliensis polysaccharides induce a very promising immunostimulation effect in vivo and in vitro. Therefore, it should be explored as a novel natural functional food additive.
Assuntos
Agaricus/química , Extratos Celulares/farmacologia , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Polissacarídeos/farmacologia , Fatores Etários , Animais , Extratos Celulares/isolamento & purificação , Citocinas/biossíntese , Feminino , Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/administração & dosagem , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Fagocitose/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Polissacarídeos/isolamento & purificação , Células RAW 264.7 , Baço/efeitos dos fármacos , Baço/imunologia , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
Ornithodoros brasiliensis is a nidicolous tick only found in the southern Brazilian highlands region. O. brasiliensis parasitism is frequently associated with toxicosis syndrome, which can lead to severe reactions, ranging from local pruritus and pain to systemic disturbances both in humans and dogs. One of the most frequent findings associated with an O. brasiliensis bite is a slow healing lesion at the site of tick attachment, which can take several weeks to heal. This work tested the hypothesis that an O. brasiliensis salivary gland homogenate is able to modulate the skin wound-healing process in vivo, using a model of excisional skin lesion in rats, which are divided into two groups: (1) control group and (2) treated group, which topically received salivary gland homogenate equivalent to the protein amount of one whole salivary gland (≈5 µg protein). The hypothesis that O. brasiliensis salivary gland homogenates interfere with endothelial cell proliferation, a key role phenomenon in wound healing, was also tested. O. brasiliensis salivary gland homogenates significantly delay skin wound healing. The time to full healing of skin lesions in control rats was 15 days, contrasting with 24 days in rats topically treated with O. brasiliensis salivary gland homogenates. The calculated HT50 (healing time to recover 50% of the wound area) for control groups was 3.6 days (95% CI, 3.2-3.9) and for salivary gland treated rats was 7.7 days (95% CI, 7.0-8.4). Salivary gland homogenates have a strong cytotoxic activity on cultured endothelial cells (LC50, 13.6 mg/ml). Also, at sublethal concentrations (≤3 mg/ml), salivary gland homogenates have a remarkable anti-proliferative activity (IC50 0.7 mg/ml) on endothelial cells, equivalent to ≈0.03 salivary gland pairs, an activity which seems to be much greater than reported for any other tick species. This is the first report about the biological activities of O. brasiliensis salivary compounds and provides the first in vivo evidence to support the concept of wound-healing modulation by tick salivary secretions. Results shown here contribute to an understanding of O. brasiliensis tick toxicosis syndrome, and also increase our knowledge of tick salivary bioactive compounds.
Assuntos
Extratos Celulares/toxicidade , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Ornithodoros/química , Cicatrização/efeitos dos fármacos , Animais , Extratos Celulares/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Masculino , Ratos , Ratos Wistar , Glândulas Salivares/química , Pele/lesõesRESUMO
This study analyzed the histopathology of rabbit skin, previously immunized with SGE2, SGE4, and SGE6 gland extracts prepared from salivary glands of Rhipicephalus sanguineus female with 2, 4, and 6 days of feeding, at the region of the R. sanguineus female feeding lesion 2, 4, and 6 days after tick attachment. In this work, infestation-naïve New Zealand White rabbits were inoculated either with the extracts (test group (TG)) or with phosphate buffer and complete Freund's adjuvant mixture (control group 2 (CG2)). Each extract-inoculated- (TG and CG2) and non-inoculated (CG1) rabbit was subsequently infested with R. sanguineus. Skin biopsies were collected from the rabbit at the tick feeding lesion at 2, 4, and 6 days of feeding. Results revealed that rabbit immunization with gland extracts induced acquisition of resistance against this species. It should be stated that the SGE4 extract was the most effective in developing an immune-inflammatory response against ectoparasites, being this process characterized by the presence of an early and intense inflammatory cell infiltrate. On the other hand, SGE6 extract caused a later appearance of resistance with less infiltrate occurrence and intense edema at the feeding lesion site. As to the inflammatory process deriving from SGE2 extract inoculation, it was the less intense. It was concluded that immunization with different extracts from R. sanguineus female salivary glands did not change microscope features of the inflammatory process, although an earlier or more intense and later response, which was also dependent on the inoculate extract, was noticed.
Assuntos
Extratos Celulares/imunologia , Ectoparasitoses/imunologia , Ectoparasitoses/patologia , Mordeduras e Picadas de Insetos/patologia , Proteínas de Insetos/imunologia , Rhipicephalus sanguineus/imunologia , Pele/patologia , Animais , Biópsia , Extratos Celulares/isolamento & purificação , Feminino , Histocitoquímica , Proteínas de Insetos/isolamento & purificação , Microscopia , Coelhos , Glândulas Salivares/química , Vacinação/métodosRESUMO
BACKGROUND: Chemotherapy for leishmaniasis, a disease caused by Leishmania parasites, is expensive and causes side effects. Furthermore, parasite resistance constitutes an increasing problem, and new drugs against this disease are needed. In this study, we examine the effect of the compound 8,10,18-trihydroxy-2,6-dolabelladiene (Dolabelladienetriol), on Leishmania growth in macrophages. The ability of this compound to modulate macrophage function is also described. METHODOLOGY/PRINCIPAL FINDINGS: Leishmania-infected macrophages were treated with Dolabelladienetriol, and parasite growth was measured using an infectivity index. Nitric oxide (NO), TNF-α and TGF-ß production were assayed in macrophages using specific assays. NF-kB nuclear translocation was analyzed by western blot. Dolabelladienetriol inhibited Leishmania in a dose-dependent manner; the IC(50) was 44 µM. Dolabelladienetriol diminished NO, TNF-α and TGF-ß production in uninfected and Leishmania-infected macrophages and reduced NF-kB nuclear translocation. Dolabelladienetriol inhibited Leishmania infection even when the parasite growth was exacerbated by either IL-10 or TGF-ß. In addition, Dolabelladienetriol inhibited Leishmania growth in HIV-1-co-infected human macrophages. CONCLUSION: Our results indicate that Dolabelladienetriol significantly inhibits Leishmania in macrophages even in the presence of factors that exacerbate parasite growth, such as IL-10, TGF-ß and HIV-1 co-infection. Our results suggest that Dolabelladienetriol is a promising candidate for future studies regarding treatment of leishmaniasis, associated or not with HIV-1 infection.
Assuntos
Antiprotozoários/farmacologia , Extratos Celulares/farmacologia , Diterpenos/farmacologia , Leishmania/efeitos dos fármacos , Phaeophyceae/química , Animais , Antiprotozoários/isolamento & purificação , Extratos Celulares/isolamento & purificação , Células Cultivadas , Diterpenos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Leishmania/crescimento & desenvolvimento , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Testes de Sensibilidade Parasitária , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: An aqueous extract and fraction from the marine sponge Petromica citrina have antibacterial activity. We performed a chemical and biological characterization of the antibiotic substance from P. citrina and investigated its mode of action on Staphylococcus aureus cells. METHODS: The inhibitory activity of the aqueous extract of P. citrina was determined against 14 bacteria belonging to type strains and clinical antibiotic-resistant strains. The aqueous extract was fractionated under bioassay guidance and the bioactive substance was identified by its (1)H-NMR, (13)C-NMR and mass spectra. The MIC and the MBC of this substance were determined. This substance was also subjected to cytotoxic bioassays. The mode of action on S. aureus cells was investigated by light and transmission electron microscopy analysis. RESULTS: P. citrina showed a large spectrum of activity against type strains and resistant-bacteria such as S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum and Neisseria gonorrhoeae. The aqueous extract was fractionated and halistanol trisulphate (24ε,25-dimethylcholestane-2ß,3α,6α-triol trisodium sulphate) was isolated for the first time from P. citrina. Halistanol trisulphate had a bactericidal effect on exponentially growing S. aureus cells at the MIC (512 mg/L). Cytotoxicity biossays showed moderate toxicity against cancer cell line L929 (fibrosarcoma). This substance apparently acts by damaging the cell membrane, with subsequent cell lysis. CONCLUSIONS: Halistanol trisulphate is a broad-spectrum antibiotic isolated from P. citrina with a mode of action involving disruption of the cytoplasmic membrane. It is a new candidate for research on antibacterial substances.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Poríferos/química , Esteróis/isolamento & purificação , Esteróis/farmacologia , Animais , Antibacterianos/química , Bactérias/citologia , Extratos Celulares/química , Extratos Celulares/isolamento & purificação , Extratos Celulares/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fracionamento Químico , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , MicroscopiaRESUMO
El propósito del trabajo fue determinar si el extracto de Staphylococcus aureus in vitro puede modificar la quimiocinesis e inducir la quimiotaxis de las células polimorfonucleares de la sangre periférica en donadores sanos. Se determinó la quimiocinesis y la quimiotaxis de los polimorfonucleares de la sangre periférica de 30 donadores sanos de uno y otro sexo con un límite de edad entre 18 y 40 años. Se les extrajeron 5 mL de sangre periférica separando los polimorfonucleares por el método de Boyum y se retaron con extracto de Staphylococcus aureus y C5a como quimiotácticos, y solución de Hank para medir quimiocinesis. Esta tuvo un promedio de 54.6ñ8.8 µm, la respuesta quimiotáctica a C5a fue 89ñ12.5 µm y con el extracto bacteriano fue 103ñ20.1 µm (p<0.001. Análisis estadístico: prueba de Wilcoxon. Se concluye que el extracto completo de Staphylococcus aureus estímula in vitro la quimiotaxis de polimorfonucleares de donadores sanos y que esta estimulación es comparable con lo quimioatrayentes conocidos como C5a