Helena Lenasi

Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

Effect of Regular Physical Training on Cutaneous Microvascular Reactivity

PURPOSE To investigate whether regular intense physical training induces changes in microvascular reactivity of human glabrous and nonglabrous skin. METHODS Subjects were physically trained competitive cyclists (N = 19) and age-matched sedentary controls (N = 20). We measured cutaneous microvascular blood flow on the dorsum of the hand (nonglabrous skin) and on the finger pulp (glabrous skin) using the laser-Doppler (LD) method. Endothelium-dependent vasodilation was assessed by an iontophoretic application of acetylcholine (ACh) on the dorsum of the hand and by an induction of postocclusive reactive hyperemia (PRH) on the finger pulp. Endothelium-independent vasodilation was assessed on the dorsum of the hand by iontophoretically applied sodium-nitroprusside (SNP). RESULTS The ACh-evoked increase in LD flux (LDF) was significantly greater in the group of cyclists (7.5-fold +/- 0.5 increase) as compared with controls (5.9-fold +/- 0.5). We found no differences in the peak LDF during PRH, whereas the recovery time of PRH was significantly longer (241.5 +/- 21.6 s in trained vs 154.6 +/- 9.3 s in sedentary group) and the area under the PRH curve significantly larger in the group of trained subjects (19,066 +/- 2,653 PU x s in trained vs 12,168 +/- 864 PU x s in sedentary). In contrast, we found a significantly smaller response to SNP in the group of cyclists (6.2-fold +/- 0.5 increase) as compared with sedentary subjects (7.8-fold +/- 0.5 increase). CONCLUSION The results of our study point to a greater vasodilator capacity of endothelium in glabrous as well as in nonglabrous skin in the group of physically trained subjects. In addition, our results indicate that regular physical activity also modifies the reactivity of vascular smooth muscle cells.

Novel minimally invasive laser treatment of urinary incontinence in women

Urinary incontinence (UI) is a common disorder that affects women of various ages and impacts all aspects of life. Our aim was to evaluate the non‐invasive erbium:yttrium‐aluminum‐garnet (Er:YAG) laser that exploits its thermal effect and has been used in reconstructive and rejuvenation surgery as a potential treatment strategy for stress UI (SUI) and mixed UI (MUI).

Regular physical activity alters the postocclusive reactive hyperemia of the cutaneous microcirculation

Regular physical activity leads to increased endothelium-dependent vasodilatation. Postocclusive reactive hyperemia (PRH) is a transient increase of blood flow after the release of an arterial occlusion and has been used as a clinical tool to estimate endothelial function. The aim of our study was to assess the potential effect of regular physical training on PRH of skin microcirculation. Skin blood flux was estimated by laser-Doppler fluxmetry (LDF) in two groups of subjects: 12 highly trained athletes and 12 age-matched sedentary controls. LDF was measured on two specific skin sites: volar aspect of the forearm (nonglabrous area) and finger pulp of the middle finger (glabrous area). After the release of a 3-min occlusion of the brachial artery, we determined the following indices of PRH: the time to peak (tpeak), the maximal LDF (LDFpeak), the recovery time (trec), the area under the PRH curve (AUC). Baseline LDF did not differ between the trained and sedentary subjects in either site. On the forearm, we found no significant differences in either PRH parameter. On the contrary, on the finger pulp, there were statistically significant differences in the tpeak and the AUC (p < or = 0.05). The results show an altered PRH response of skin microcirculation in the finger pulp in the trained subjects. We may speculate that this could be the result of an increased endothelial vasodilator capacity. Further, the potential adaptations of the endothelium differ between the glabrous and nonglabrous skin sites.

The role of nitric oxide- and prostacyclin-independent vasodilatation in the human cutaneous microcirculation: effect of cytochrome P450 2C9 inhibition.

The component of the flow‐ or agonist‐dependent vasodilatation, insensitive to inhibitors of nitric oxide (NO) synthases (NOS) or cyclooxygenases (COX), is suggested to reflect the production of an endothelium‐dependent hyperpolarizing factor (EDHF). The identity of EDHF in humans remains controversial; in coronary arterioles, it appears to be a cytochrome P450 (CYP) 2C9‐derived metabolite, whereas there are no data for human skin microcirculation. Therefore, the aim of our study was to investigate the role of the NO‐ and prostacyclin (PGI2)‐independent mechanism, particularly the potential involvement of CYP 2C9, in skin microcirculation. We measured skin blood flow on the volar aspect of the forearm in 12 healthy subjects by laser‐Doppler fluxmetry (LDF). The inhibitors of NOS, Nω‐monomethyl‐L‐arginine (L‐NMMA), and cyclooxygenase (COX), diclofenac, as well as sulfaphenazole, the specific CYP 2C9 inhibitor, and saline as control, were administered to the measurement sites by an intradermal microinjection in different combinations. Afterwards, baseline LDF was assessed and iontophoresis of acetycholine (ACh) applied. Combined NOS and COX inhibition had no effect on baseline LDF, whereas it significantly reduced the ACh‐induced increase in LDF (t‐test, P<0·05). Sulfaphenazole did not affect baseline LDF either in the control site or in the L‐NMMA‐ and diclofenac‐pretreated site. In addition, sulfaphenazole did not attenuate the ACh‐induced vasodilatation in either site. We conclude that a NO‐ and PGI2‐independent vasodilator mechanism, potentially attributable to EDHF, contributes substantialy to the ACh‐induced vasodilatation in human skin microcirculation and that it is probably not a CYP 2C9‐derived metabolite.

Identification of progesterone binding sites in the plasma membrane of the filamentous fungus Cochliobolus lunatus

Plasma membrane associated binding sites for progesterone have been identified in the filamentous fungus Cochliobolus lunatus (C. lunatus). The Kd for progesterone determined by Scatchard analysis was 13.9 +/- 5.7 nM and the Bmax was 250-360 fmol/mg protein. A broad ligand specificity of these binding sites is suggested by the observation that all tested steroids, regardless of their capability to act as inducers of the 11 beta-steroid hydroxylase, competed at 250-fold excess with [3H]progesterone binding. A biological role of these plasma membrane associated steroid binding sites is nevertheless suggested since in protoplasts which were devoid of them, 11 beta-steroid hydroxylase could not be induced. Progesterone binding sites were present in the plasma membrane as well as in the cytosol and were detected in this fraction, in contrast to the plasma membrane fraction, only under special experimental conditions in respect to redox state. Kd and Bmax of cytosol binding sites were of the same order of magnitude compared to the plasma membrane progesterone binding sites. Ethisterone and 4-cholesten-3-one which cannot induce 11 beta-hydroxylase competed efficiently for plasma membrane binding sites; ethisterone, however also competed for cytosol binding sites and acted, in contrast with 4-cholesten-3-one, as antagonist in the induction of 11 beta-steroid hydroxylase in C. lunatus. On the basis of presented evidence we concluded that C. lunatus contains binding sites for steroids in the plasma membrane and in the cytosol and that both types of binding site are involved in the process of induction of enzymes which transform steroids in this fungus.

Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans

Rhizopus nigricans (R. nigricans) transforms fungitoxic progesterone into the less toxic 11alpha-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC(50) values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40nM ((3)H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC(50) of progesterone 2.3 +/- 0.1 x 10(-7)M, EC(50) of androsten-3,17-dione 24 +/- 2 x 10(-7)M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons alpha-naphthoflavone and ketoconazole exhibited EC(50) values of 0.3 +/- 0.01 x 10(-7)M and 27 +/- 5 x 10(-7)M, respectively, whereas beta-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of alpha-naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.

The response of filamentous fungus Rhizopus nigricans to flavonoids

The saprophytic fungus Rhizopus nigricans constitutes a serious problem when thriving on gathered crops. The identification of any compounds, especially natural ones, that inhibit fungal growth, may therefore be important. During its life cycle, Rhizopus nigricans encounters many compounds, among them the flavonoids, plant secondary metabolites that are involved in plant defense against pathogenic microorganisms. Although not being a plant pathogen, Rhizopus nigricans may interact with these compounds in the same way as plant pathogens – in response to the fungitoxic effect of flavonoids the fungi transform them into less toxic metabolites. We have studied the interaction of R. nigricans with some flavonoids. Inhibition of hyphal spreading (from 3% to 100%) was observed by 300 μM flavones, flavanones and isoflavones, irrespective of their basic structure, oxidized or reduced C‐ring, and orientation of the B‐ring. However, a hydrophobic A‐ring was important for the toxicity. R. nigricans transformed some of the flavonoids into glucosylated products. Recognition of substrates for glucosylating enzyme(s) did not correlate with their fungitoxic effect but depended exclusively on the presence of a free –OH group in the flavonoid A‐ring and of a hydrophobic B‐ring. Although the fungus produced glucosyltransferase constitutively, an additional amount of the enzyme was induced by the substrate flavonoid. Moreover, effective detoxification was shown to require the presence of glucose. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

G-Protein coupled progesterone receptors in the plasma membrane of fungus Rhizopus nigricans

Abstract We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum.

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15alpha-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein beta subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Deltafgb1 strain, suggesting a role for the G-protein beta subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Deltafgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.