RESUMO

Hypertension augments while exercise training corrects the increased vesicle trafficking (transcytosis) across the blood-brain barrier (BBB) within preautonomic areas and the autonomic imbalance. There is no information on a possible mechanism(s) conditioning these effects. Knowing that Mfsd2a is the major transporter of docosahexaenoic acid (DHA) and that Mfsd2a knockout mice exhibited leaky BBB, we sought to identify its possible involvement in hypertension- and exercise-induced transcytosis across the BBB. Spontaneously hypertensive rats (SHR) and Wistar rats were submitted to treadmill training (T) or kept sedentary (S) for 4 wk. Resting hemodynamic/autonomic parameters were recorded in conscious chronically cannulated rats. BBB permeability within the hypothalamic paraventricular nucleus (PVN) was evaluated in anesthetized rats. Brains were harvested for Mfsd2a and caveolin-1 (an essential protein for vesicle formation) expression. SHR-S versus Wistar-S exhibited elevated arterial pressure (AP) and heart rate (HR), increased vasomotor sympathetic activity, reduced cardiac parasympathetic activity, greater pressure variability, reduced HR variability, and depressed baroreflex control. SHR-S also showed increased BBB permeability, reduced Mfsd2a, and increased caveolin-1 expression. SHR-T versus SHR-S exhibited increased Mfsd2a density, reduced caveolin-1 protein expression, and normalized PVN BBB permeability, which were accompanied by resting bradycardia, partial AP drop, reduced sympathetic and normalized cardiac parasympathetic activity, increased HR variability, and reduced pressure variability. No changes were observed in Wistar-T versus Wistar-S. Training is an efficient tool to rescue Mfsd2a expression, which by transporting DHA into the endothelial cell reduces caveolin-1 availability and vesicles' formation. Exercise-induced Mfsd2a normalization is an important mechanism to correct both BBB function and autonomic control in hypertensive subjects.

Assuntos

RESUMO

BACKGROUND: The development of multidrug resistance (MDR) associated with the overexpression of the efflux transporters Mdr1 and Cdr1 in Candida species impedes antifungal therapies. The urgent need for novel agents able to inhibit the function of both pumps, led us to evaluate this property in 137 extracts obtained from Argentinian plants. METHODS: The ability of the extracts to reverse efflux pump-mediated MDR was determined with an agar chemosensitization assay using fluconazole (FCZ) resistant Mdr1- and Cdr1-overexpressing clinical isolates of Candida albicans and Candida glabrata as well as Saccharomyces cerevisiae strains selectively expressing Mdr1 (AD/CaMDR1) or Cdr1 (AD/CaCDR1). The resistance-reversing activity of the most potent extracts was further confirmed using a Nile Red accumulation assay. RESULTS: Fifteen plant extracts overcame the FCZ resistance of Candida albicans 1114, which overexpresses CaMdr1 and CaCdr1, and AD/CaMDR1, with those from Acalypha communis and Solanum atriplicifolium being the most effective showing 4- to 16-fold reversal of resistance at concentrations ≥ 25 µg/mL. Both extracts, and to a lesser extent that from Pterocaulon alopecuroides, also restored FCZ sensitivity in CgCdr1-overexpressing C. glabrata 109 and in AD/CaCDR1 with fold reversal values ranging from 4 to 32 and therefore demonstrating a dual effect against Mdr1 and Cdr1. Both, A. communis and S. atriplicifolium extracts at concentrations ≥ 12.5 and ≥ 25 µg/mL, respectively, increased the intracellular Nile Red accumulation in all yeast strains overexpressing efflux pumps. CONCLUSIONS: The non-toxic and highly active extracts from A. communis and S. atripicifolium, provide promising sources of compounds for potentiating the antifungal effect of FCZ by blocking the efflux function of Mdr1 and Cdr1 transporters.

Assuntos

RESUMO

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.

Assuntos

RESUMO

JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins.

RESUMO

In the struggle for life, the capacity of microorganisms to synthesize and secrete toxic compounds (inhibiting competitors) plays an important role in successful survival of these species. This ability must come together with the capability of being unaffected by these same compounds. Several mechanisms are thought to avoid the toxic effects. One of them is toxin extrusion from the intracellular environment to the outside vicinity, using special transmembrane proteins, referred to as transporters. These proteins are also important for other reasons, since most of them are involved in nutrient uptake and cellular excretion. In cancer cells and in pathogens, and particularly in fungi, some of these proteins have been pointed out as responsible for an important phenotype known as multidrug resistance (MDR). In the present study, we tried to identify in the Paracoccidioides brasiliensis transcriptome, transporter-ortholog genes from the two major classes: ATP binding cassette and major facilitator superfamily transporter. We found 22 groups with good similarity with other fungal ATP binding cassette transporters, and four Paracoccidioides brasilienses assembled expressed sequence tags that probably code for major facilitator superfamily proteins. We also focused on fungicide resistance orthologs already characterized in other pathogenic fungi. We were able to find homologs to C. albicans CDR1, CDR2, and MDR1, Saccharomyces cerevisiae PDR5 and Aspergillus AtrF genes, all of them related to azole resistance. As current treatment for paracoccidioidomycosis mainly uses azole derivatives, the presence of these genes can be postulated to play a similar role in P. brasiliensis, warning us for the possibility of resistant isolate emergence.

Assuntos