Production, purification and characterization of laccase from pleurottus ostreatus grown on tomato pomace

A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of 2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices and it was purified on a Sepharose 6B-BDGE-urea column with final enzyme recoveries of about 60%, specific activity of 6.0 and 18.0 U/mg protein and purification factors in the range of 14-46. It was also possible to demonstrate that metal-free laccase did not adsorb to Sepharose 6B-BDGE-urea column which suggests that adsorption of native laccase on this affinity matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II) ions of laccase. The kinetic parameters (V (max), K (m) , K (cat), and K (cat)/K (m) ) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand.

In vitro susceptibility of Paracoccidioides brasiliensis yeast form to antifungal agents

A study was conducted to determine the susceptibility of P. brasiliensis yeast form to amphotericin B (A), ketoconazole (K), 5-fluorocytosine (5-FC) and rifampin (R). The three isolates tested produced minimal inhibitory concentrations (MICs) (mcg/ml) in the following range: A: 0.09-0.18; K: 0.001-0.007; 5-FC: 62.5-250 and R: 40-80. The minimal fungicidal concentrations (MFC) were several times higher than the corresponding MICs. Precise MFC for 5-FC were not obtained (> 500 mcg/ml). Combination of K plus A proved synergic, with the fractional inhibitory concentration (FIC) indices revealing synergy when the drugs were combined at the 1 to 1 and 1 to 5 MIC ratios. R (40 mcg/ml) appeared to antagonize K. These results indicate promise for the combined use of K plus A as a therapeutical regimen.

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast phaffia rhodozyma and its sexual form, xanthophyllomyces dendrorhous

Applied and Environmental Microbiology, Vol. 73, No.4

Immunoglobulins and C3 in the P. brasiliensis granuloma

The experimental model of paracoccidioidomycosis induced in mice by the intravenous injection of yeast-forms of P. brasiliensis (Bt2 strain; 1 x 10(6) viable fungi/animal) was used to evaluate sequentially 2, 4, 8, 16 and 20 weeks after inoculation: 1. The presence of immunoglobulins and C3 in the pulmonary granuloma-ta, by direct immunofluorescence; 2. The humoral (immunodiffusion test) and the cellular (footpad sweeling test) immune response; 3. The histopathology of lesions. The cell-immune response was positive since week 2, showing a transitory depression at week 16. Specific antibodies were first detected at week 4 and peaked at week 16. At histology, epithelioid granulomas with numerous fungi and polymorphonuclear agreggates were seen. The lungs showed progressive involvement up to week 16, with little decrease at week 20. From week 2 on, there were deposits of IgG and C3 around fungal walls within the granulomas and IgG stained cells among the mononuclear cell peripheral halo. Interstitital immunoglobulins and C3 deposits in the granulomas were not letected. IgG and C3 seen to play an early an important role in. the host defenses against P. brasiliensis by possibly cooperating in the killing of parasites and blocking the antigenic diffusion.

Radiometric detection of metabolic activity of Paracoccidioides brasiliensis and its susceptibility to amphotericin B and diethylstilbestrol

Paracoccidioidomycosis (South American blastomycosis) is a systemic disease, strikingly more frequent in males, caused by the dimorphic fungus Paracoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on this fungus "in vitro". The Y form of the yeast, grown in liquid Sabouraud medium was inoculated into sterile reaction vials containing the 6B aerobic medium along with 2.0 μCi of 14C-substrates. Control vials, prepared in the same way, contained autoclaved fungi. To study the effects of amphotericin B (AB) (0.1 and 10 μg/ml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 μg/ml) extra controls with live fungi and no drug were used. All vials were incubated at 35°C and metabolism measured daily with a Bactec instrument. 14CO2 production by P. brasiliensis was slow and could be followed for as long as 50 days. AB at 10mg/ml and DSB at 5 μg/ml inhibited the metabolism and had a cidal effect on this fungus. The results with DSB might explain the low incidence of the disease in females. This technique shows promise for studying metabolic pathways, investi gating more convenient 14C-substrates to expedite radiometric detection and for monitoring the effects of other drugs and factors on the metabolism of P. brasiliensis "in vitro".

Inapparent lung involvement in patients with the subacute juvenile type of paracoccidioidomycosis

Three patients with the diagnosis of subacute juvenile paracoccidioidomycosis who, at the time of their first visit, had no signs or symptoms of lung involvement, were studied. Initially the diagnosis was confirmed by the observation of P. brasiliensis in biopsy material obtained from clinically involved lymphadenopathies. The lung X-rays done in all patients, did not reveal pathologic changes, although it was possible to observe and isolate the fungus from sputum samples obtained from the three patients. This fact reinforces the pulmonary genesis of the mycosis and proofs the existence of a pulmonary primary infection, even in patients with the juvenile manifestations, in whom the lung component is obscured by the predominant lymph node involvement.

Deep crustal structure across a young passive margin from wide-angle and reflection seismic data (The Sardinia Experiment) - I. Gulf of Lion's margin

The conjugate margins system of the Gulf of Lion and West Sardinia (GLWS) represents a unique natural laboratory for addressing fundamental questions about rifting due to its landlocked situation, its youth, its thick sedimentary layers, including prominent palaeo-marker such as the MSC event, and the amount of available data and multidisciplinary studies. The main goals of the SARDINIA experiment, were to (i) investigate the deep structure of the entire system within the two conjugate margins: the Gulf of Lion and West Sardinia, (ii) characterize the nature of the crust, and (iii) define the geometry of the basin and provide important constrains on its genesis. This paper presents the results of P-wave velocity modelling on three coincident near-vertical reflection multi-channel seismic (MCS) and wide-angle seismic profiles acquired in the Gulf of Lion, to a depth of 35 km. A companion paper [part II Afilhado et al., 2015] addresses the results of two other SARDINIA profiles located on the oriental conjugate West Sardinian margin. Forward wide-angle modelling of both data sets confirms that the margin is characterised by three distinct domains following the onshore unthinned, 33 km-thick continental crust domain: Domain I is bounded by two necking zones, where the crust thins respectively from 30 to 20 and from 20 to 7 km over a width of about 170 km; the outermost necking is imprinted by the well-known T-reflector at its crustal base; Domain II is characterised by a 7 km-thick crust with anomalous velocities ranging from 6 to 7.5 km/s; it represents the transition between the thinned continental crust (Domain I) and a very thin (only 4-5 km) "atypical" oceanic crust (Domain III). In Domain II, the hypothesis of the presence of exhumed mantle is falsified by our results: this domain may likely consist of a thin exhumed lower continental crust overlying a heterogeneous, intruded lower layer. Moreover, despite the difference in their magnetic signatures, Domains II and III present the very similar seismic velocities profiles, and we discuss the possibility of a connection between these two different domains.

Drug susceptibility of Plasmodium falciparum in the western Amazon region, state of Acre, Brazil

Field studies in the western Amazon region (state of Acre, Brazil) indicate that the 4-aminoquinolines, as well as the combined regimen with sulfadoxine-pyrimethamine, can no longer be recomended for the treatment and prophylaxis of P. falciparum infections in this region. Quinine remains an effective drug when used correctly. However, compliance problems arise due to the often occurring side-effects during a ten day regimen. Prospects of overcoming these constraints by combining a short course of quinine with other drugs are limited, because of the lack of suitable partner compounds. For this reason quinine/clindamycin appears to be a more practical therapy of P. falciparum malaria. In vitro data from this study suggest that mefloquine is another effective alternative for the treatment of falciparum malaria in this Amazon region.

Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress

The impact of metals (Cd, Cr, Cu and Zn) on growth, cell volume and cell division of the freshwateralga Pseudokirchneriella subcapitata exposed over a period of 72 h was investigated. The algal cells wereexposed to three nominal concentrations of each metal: low (closed to 72 h-EC10values), intermediate(closed to 72 h-EC50values) and high (upper than 72 h-EC90values). The exposure to low metal concen-trations resulted in a decrease of cell volume. On the contrary, for the highest metal concentrations anincrease of cell volume was observed; this effect was particularly notorious for Cd and less pronouncedfor Zn. Two behaviours were found when algal cells were exposed to intermediate concentrations ofmetals: Cu(II) and Cr(VI) induced a reduction of cell volume, while Cd(II) and Zn(II) provoked an oppositeeffect. The simultaneous nucleus staining and cell image analysis, allowed distinguishing three phases inP. subcapitata cell cycle: growth of mother cell; cell division, which includes two divisions of the nucleus;and, release of four autospores. The exposure of P. subcapitata cells to the highest metal concentrationsresulted in the arrest of cell growth before the first nucleus division [for Cr(VI) and Cu(II)] or after thesecond nucleus division but before the cytokinesis (release of autospores) when exposed to Cd(II). Thedifferent impact of metals on algal cell volume and cell-cycle progression, suggests that different toxic-ity mechanisms underlie the action of different metals studied. The simultaneous nucleus staining andcell image analysis, used in the present work, can be a useful tool in the analysis of the toxicity of thepollutants, in P. subcapitata, and help in the elucidation of their different modes of action.

Spectral invariants of periodic nonautonomous discrete dynamical systems

For an interval map, the poles of the Artin-Mazur zeta function provide topological invariants which are closely connected to topological entropy. It is known that for a time-periodic nonautonomous dynamical system F with period p, the p-th power [zeta(F) (z)](p) of its zeta function is meromorphic in the unit disk. Unlike in the autonomous case, where the zeta function zeta(f)(z) only has poles in the unit disk, in the p-periodic nonautonomous case [zeta(F)(z)](p) may have zeros. In this paper we introduce the concept of spectral invariants of p-periodic nonautonomous discrete dynamical systems and study the role played by the zeros of [zeta(F)(z)](p) in this context. As we will see, these zeros play an important role in the spectral classification of these systems.

Paracoccidioides brasiliensis: a mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus)

A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135, has got all the characteristics of P. brasiliensis, with a strong antigenic power and low virulence for guinea-pigs and Wistar rats. The specific exoantigen of P. brasiliensis - the glycoprotein with a molecular weight of 43 kDa - was easily demonstrated with double immunodiffusion, immunoelectrophoresis, SDS-PAGE and immunobloting techniques.

Evaluation of the allergenicity of spore and mycelia extracts of Pisolithus tinctorius

The antigenic and allergenic chemical analysis of spore and mycelia extracts of Pisolithus tinctorius was carried out. The spores were collected from basidiocarps in plantations of Eucalyptus spp and the mycelia from culture in MNM medium. With basis on the fungus growth curve, the mycelia masses were obtained after 10, 20, 30, and 40 days of incubation, which correspond, respectively, to the beginning, middle and end of the log phase, and beginning of the decline phase. The mycelia masses, together with the spores, were submitted to the action of three extractors (Coca, Tris-HCl, and ammonium bicarbonate). The contents of carbohydrates and proteins were determined. The SDS-PAGE electrophoretical analysis revealed separate fractions in these extracts, besides common fractions, in function of cultivation time and extraction methods. The selected extracts for the allergic tests were the ones with the highest number of fractions. The prick-tests were conducted in 374 patients – rural workers, eucalyptus plantation works and college students. The positivity to the "prick test" with the antigenic extract of P. tinctorius was, respectively, 3.78%, 28.20% and 6.40%. Most prick-test positive patients (82.75%) also presented symptoms of respiratory allergy (asthma and rhinitis). There was no reactivity difference when the spore and mycelia extracts were employed. The analysis of the positive patients’ sera revealed the presence of IgE specific to the P. tinctorius antigens. Since Pisolithus tinctorius is found as mycorrhyza of Eucalyptus spp, and this plant is used in reforestation in most countries, the importance of that fungus should be regarded as a possible cause of respiratory allergies, especially in occupationally exposed workers

Paracoccidioidomycosis in children in the state of Rio de Janeiro (Brazil): Geographic distribution and the study of a "reservarea"

Thirty six cases of acute disseminated paracoccidioidomycosis in 3 to 12 year-old children, natives of the state of Rio de Janeiro, were seen in the period 1981-1996. All patients were residents in the rural region of 15 counties, scattered on the Southwestern part of this state. The rural region of two neighboring counties, where 16 cases (44.4%) occurred, was visited. It exhibited the environmental conditions that are considered favorable to the survival of P. brasiliensis. The most important of these conditions, abundant watercourses and autochthonous forest, are distributed on well defined and limited areas, in which the dwellings are also localized. Probably, a careful epidemiological study of forthcoming cases of the disease in children may facilitate the search for the micro-niche of the fungus.

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20oC up to 10 days or at -70oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

Penicillium glabrum cork colonising isolates – preliminary analysis of their genomic similari

Cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are yet to be completely understood and are considered to be involved in the so called “cork taint” of bottled wine. It is essential to identify environmental constraints which define the appearance of the colonising fungal species and to trace their origin to the forest and/or as residents in the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic biologic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was identified, followed by a high diversity of Penicillium species. Penicillium glabrum, found in all samples, was the most frequent isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork resident fungi can also contrib