Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles

Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis , four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.     

VDAC1 is a transplasma membrane NADH-ferricyanide reductase

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.     

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.     

Thyrotropin secretagogues reduce rat pituitary neuromedin B, a local thyrotropin release inhibitor

Neuromedin B (NB), a bombesin-like peptide, highly concentrated in rat pituitary gland, has been shown to act as an autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here it is shown that a single injection of thyrotropin-releasing hormone (TRH, 1.5 microg/animal, ip), the most important stimulator of thyrotropin secretion, induced approximately 35%-45% decrease in pituitary NB content in rats, as well as an important decrease in NB mRNA at 15 and 30 min (P < 0.05). Acute cold exposure, which induced higher serum TSH with a peak at 30 min, was associated with progressive decrease in pituitary NB, starting at 15 min although only reaching statistical significance after 2 hr (P < 0.05). Although not involved in the early peak, the decrease in NB may be contributing to maintenance of higher serum TSH in cold-exposed animals compared with those at room temperature. Fed rats, 2 hr after being subcutaneously injected with mouse recombinant leptin (8 microg /100 g body wt), showed a x2 increase in serum TSH and 38% reduction in pituitary NB (P < 0.05). In conclusion, TRH and leptin rapidly decreased pituitary NB and it is first proposed that the reduction of the inhibitory tonus of NB on TSH release will ultimately contribute to the amplification of TSH secretion elicited by TSH secretagogues.     

Can Phlorotannins Purified Extracts Constitute a Novel Pharmacological Alternative for Microbial Infections with Associated Inflammatory Conditions?

Bacterial and fungal infections and the emerging multidrug resistance are driving interest in fighting these microorganisms with natural products, which have generally been considered complementary to pharmacological therapies. Phlorotannins are polyphenols restricted to brown seaweeds, recognized for their biological capacity. This study represents the first research on the antibacterial, antifungal, anti-inflammatory and antioxidant activity of phlorotannins purified extracts, which were obtained from ten dominant brown seaweeds of the occidental Portuguese coast.Phlorotannins content was determined by the specific dimethoxybenzaldehyde (DMBA) method and a yield between 75 and 969 mg/Kg phloroglucinol units (dry matter) was obtained. Fucus spiralis ranked first, followed by three Cystoseira species. The anti-inflammatory potential of the purified extracts was assessed via inhibitory effect on nitric oxide (NO) production by lipopolysaccharide-stimulated RAW 264.7 macrophage cells, Cystoseira tamariscifolia being the one showing promising activity for the treatment of inflammation. NO scavenging ability was also addressed in cell free systems, F. spiralis being the species with highest capacity. The antimicrobial potential of the extracts was checked against five Gram-positive and four Gram-negative bacteria and three fungi strains, that commonly colonize skin and mucosa and are responsible for food contamination. The different extracts were more effective against Gram-positive bacteria, Staphylococcus epidermidis being the most susceptible species. Concerning antifungal activity, Trichophyton rubrum was the most sensitive species.Although the molecular mechanisms underlying these properties remain poorly understood, the results obtained turn phlorotannins purified extracts a novel and potent pharmacological alternative for the treatment of a wide range of microbial infections, which usually also present an inflammatory component. In addition to the biological properties demonstrated herein, phlorotannins extracts may also be preferred, in order to avoid side effects and allergic reactions commonly associated with synthetic drugs.     

Ubiquitination of mammalian Pex5p, the peroxisomal import receptor

Protein translocation across the peroxisomal membrane requires the concerted action of numerous peroxins. One central component of this machinery is Pex5p, the cycling receptor for matrix proteins. Pex5p recognizes newly synthesized proteins in the cytosol and promotes their translocation across the peroxisomal membrane. After this translocation step, Pex5p is recycled back into the cytosol to start a new protein transport cycle. Here, we show that mammalian Pex5p is ubiquitinated at the peroxisomal membrane. Two different types of ubiquitination were detected, one of which is thiol-sensitive, involves Cys(11) of Pex5p, and is necessary for the export of the receptor back into the cytosol. Together with mechanistic data recently described for yeast Pex5p, these findings provide strong evidence for the existence of Pex4p- and Pex22p-like proteins in mammals.     

Synthesis and potent antitumor activity of new arylamino derivatives of nor-beta-lapachone and nor-alpha-lapachone

Several arylamino derivatives of nor-beta-lapachone were synthesized in moderate to high yields and found to show very potent cytotoxicity against six neoplastic cancer cells: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast), HL-60 (leukaemia), PC-3 (prostate), and B-16 (murine melanoma), with IC(50) below 1 microg/mL. Their cytotoxicities were compared to doxorubicin and with their synthetic precursors, beta-lapachone and nor-beta-lapachone. The activity against a normal murine fibroblast L-929 showed that some of the compounds were selective against cancer cells. The absence of hemolytic activity (EC(50)>200 microg/mL), performed with erythrocyte suspensions, suggests that the cytotoxicity of the compounds was not related to membrane damage of mouse erythrocytes. For comparison purposes, one isomeric compound based on nor-alpha-lapachone was also synthesized and showed lower activity than the related ortho-derivative. The modified arylamino quinones appear as interesting new lead compounds in anti-cancer drug development.     

pH-induced conformational change of the influenza M2 protein C-terminal domain

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy, we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high- and low-pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH, although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.     

Vascular smooth muscle relaxation by a lectin from Pisum arvense: evidences of endothelial NOS pathway

The vasorelaxant effect of the lectin of Pisum arvense (PAL) seeds was investigated in rat aorta. PAL (10-100 μg/ml) was applied on aorta rings, with or without endothelium, pre-contracted with phenylephrine (Phe; 0.1 μM). Participation of endothelium derived relaxant factors was evaluated incubating the tissue with indomethacin (10 μM), L-nitro arginine methyl ester (L-NAME, 100 μM) and tetraethylammonium (TEA, 5 mM) before addition of PAL. The role of the lectin domain was investigated by addition of PAL into tissue in presence of glucose (3x 10?? M), or N-acetyl Dglucosamine (GlcNAc; 3 x 10?? M). The importance of extracellular calcium (Ca2?e) or interaction with muscarinic receptors in the relaxant effect was evaluated by addition of PAL into aorta rings containing calcium free solution (OCa) and atropine (1 μ M), respectively. PAL induced concentration-dependent relaxation in endothelized aorta (IC50 =58.38 ± 1.87 μg/ml), which was reversed by L-NAME and glucose. The lectin effect was totally inhibited when the preparation was inserted in OCa, but not in presence of atropine. Summarizing, our data showed a relaxant effect of PAL in isolated rat aorta rings in presence of endothelium, suggestive of interaction between the lectin carbohydrate binding sites with specific receptors located in vascular endothelial cells leading to nitric oxide synthase activation. This effect seems to require Ca2?e but is independent on muscarinic receptors interaction.