RESUMEN
A comparison of the diversity of bacterial communities in the larval midgut and adult gut of the European forest cockchafer (Melolontha hippocastani) was carried out using approaches that were both dependent on and independent of cultivation. Clone libraries of the 16S rRNA gene revealed 150 operational taxonomic units (OTUs) that belong to 11 taxonomical classes and two other groups that could be classified only to the phylum level. The most abundant classes were ß, δ and γ-proteobacteria, Clostridia, Bacilli, Erysipelotrichi and Sphingobacteria. Although the insect's gut is emptied in the prepupal stage and the beetle undergoes a long diapause period, a subset of eight taxonomic classes from the aforementioned eleven were found to be common in the guts of diapausing adults and the larval midguts (L2, L3). Moreover, several bacterial phylotypes belonging to these common bacterial classes were found to be shared by the larval midgut and the adult gut. Despite this, the adult gut bacterial community represented a subset of that found in the larvae midgut. Consequently, the midgut of the larval instars contains a more diverse bacterial community compared to the adult gut. On the other hand, after the bacteria present in the larvae were cultivated, eight bacterial species were isolated. Moreover, we found evidence of the active role of some of the bacterial species isolated in food digestion, namely, the presence of amylase and xylanolytic properties. Finally, fluorescence in situ hybridization allowed us to confirm the presence of selected species in the insect gut and through this, their ecological niche as well as the metagenomic results. The results presented here elucidated the heterogeneity of aerobic and facultative bacteria in the gut of a holometabolous insect species having two different feeding habits.
Asunto(s)
Escarabajos/crecimiento & desarrollo , Escarabajos/microbiología , Tracto Gastrointestinal/microbiología , Estadios del Ciclo de Vida , Metagenoma/genética , Árboles , Animales , Bacterias/clasificación , Bacterias/genética , Colorantes Fluorescentes/metabolismo , Biblioteca de Genes , Herbivoria , Larva/microbiología , Datos de Secuencia Molecular , Hojas de la Planta/parasitología , Raíces de Plantas/parasitología , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (K(i) = 5.1+/-1.4 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) were found to be positively correlated with the K(i)-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. CONCLUSIONS/SIGNIFICANCE: The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.
Asunto(s)
Antiinflamatorios/farmacología , Antineoplásicos Fitogénicos/farmacología , Curcumina/farmacología , Lactoilglutatión Liasa/antagonistas & inhibidores , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Interleucina-1beta/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lipopolisacáridos/farmacología , Modelos Biológicos , Neoplasias/patología , Fenoles/farmacología , Polifenoles , Especificidad por SustratoRESUMEN
Esters of alpha-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of alpha-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of alpha-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding alpha-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by alpha-oxo-carbonic acid esters, whilst alpha-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Citocinas/antagonistas & inhibidores , Lactatos/farmacología , Lactoilglutatión Liasa/antagonistas & inhibidores , Piruvatos/farmacología , Receptores Inmunológicos/metabolismo , Animales , Citocinas/biosíntesis , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Monocitos/efectos de los fármacos , Monocitos/enzimología , Piruvaldehído/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have isolated a gene, LdGF1, from the protozoan parasite Leishmania donovani. Overexpression of this gene confers a strong selective advantage in liquid culture after stationary phase growth arrest. We could show that recombinant L. donovani or Leishmania major, when overexpressing LdGF1, recover faster from a stationary phase growth arrest than control parasite strains. While no advantage of LdGF1 overexpression could be observed in log phase cultures or after a hydroxyurea-induced S-phase growth arrest, recovery from a cell cycle arrest due to serum deprivation was faster in LdGF1-overexpressing strains. This was found to be due to an accelerated release from a G(1) cell cycle arrest. By contrast, in a BALB/c mouse infection system, overexpression of LdGF1 in L. major resulted in reduced virulence. We conclude that increased levels of LdGF1 are beneficiary during recovery from G(1) cell cycle arrest, but pose a disadvantage inside a mammalian host. These results are discussed in the context of the observed loss of virulence during in vitro passage of Leishmania parasites.