Inhibiting the urokinase‐type plasminogen activator receptor system recovers STZ‐induced diabetic nephropathy

The urokinase‐type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR ‐(s)uPAR‐ from circulation) is to regulate podocyte function through αvβ3 integrin/Rac‐1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)‐induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvβ3 integrin/Rac‐1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ‐induced up‐regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvβ3 integrin/Rac‐1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen‐plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR‐targeting approaches.     

Efecto inhibitorio del dietilestilbestrol sobre aislamientos clínicos de Candida albicans sensibles y resistentes a los azoles

BackgroundCandida albicans is a microorganism frequently involved in several infections; the patient's oral cavity, caries niches or periodontal disease can sometimes be the reservoir.. The fungal resistance to the available treatments, among other reasons, has led to the search for new antifungal alternatives.AimsTo carry out a comparative study of the in vitro effects of diethylstilboestrol (DES) and fluconazole (FLZ) on the growth of clinical strains of C. albicans.MethodsSeven strains of C. albicans were used: a) one FLZ-sensitive culture collection strain, ATCC 90028 (ATCC); b) four oral isolates from four oncological patients with periodontal disease (period 8, 9, 10, and 11); and c) two oral isolates from an AIDS patient with oropharyngeal candidiasis: one FLZ- sensitive (2-76), and another FLZ- resistant (12-99). The MIC was evaluated by standard spectrophotometric techniques using the CLSI (M27-A3) guidelines. The inhibitory concentration 50% (IC₅₀) was calculated using functional analysis with the Graph Pad software.ResultsDES inhibited the growth of all C. albicans strains, whether sensitive or resistant to FLZ. Experimental data fitted non-linear functions of inhibitor concentration versus response. Minimum inhibitory concentrations (MIC) for DES and FLZ were as follows: 28.18 µg/ml and 4.90 µg/ml (ATCC); 17.16 µg/ml and 3.14 µg/ml (period); 27.64 µg/ml and 4.22 µg/ml (2-76); 6.16 µg/ml and 438.19 µg/ml (12-99), respectively.ConclusionsDES showed antifungal activity on all clinical C. albicans strains isolated from patients with dental and medical diseases. It showed the highest potency on the FLZ-resistant isolate.     

Efficiency and allometry of seed production in grassland forbs: site and year comparative study in the Italian Eastern Pre-Alps

Forbs are important biodiversity components of grasslands and are often threatened by management intensification. As most forbs propagate predominantly by seed, knowledge of their seed regenerative traits would improve the conservation and restoration of forb-rich ecosystems. The main seed production traits of six forbs that are common in European species-rich grasslands were studied by collecting fertile shoots from different sites and over several years. Among sites and years, variability was high, particularly in the number of inflorescences per shoot, which affected ovule production more than any other trait. Relationships between inflorescence size and the number of ovules were mainly negatively allometric or almost so, with lower flower densities in larger inflorescences. The average ovule-to-seed transformation efficiency was 58%. There was significant variation among collections of the same species, and even more between species. Species with a low ovule-to-seed transformation efficiency generally exhibited compensatory, high seed viability. Large inflorescences had high ovule to seed utilization values, probably because of better nutrient conditions. Seed germinability (average, 30%) was much lower than seed viability (average, 54%); therefore, seed dormancy was an important feature of the species studied.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.     

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.