Electrochemical and AFM characterization on gold and carbon electrodes of a high redox potential laccase from Fusarium proliferatum.

The redox potential of the T1 copper site of laccase from Fusarium proliferatum was determined by titration to be about 510 mV vs. SCE (750 mV vs. NHE), which makes it a high redox potential enzyme. Anaerobic electron transfer reactions between laccase and carbon and gold electrodes were detected, both in solution and when the enzyme was adsorbed on these surfaces. In solution, a single high-potential signal (660 mV vs. SCE) was recorded at the carbon surfaces, attributable to the T1 copper site of the enzyme. However, a well-defined oxidative process at about 660 mV and an anodic wave at 350 mV vs. SCE were recorded at the gold electrode, respectively associated with the T1 and T2 copper sites. Laccase-modified carbon electrodes behaved analogously when the enzyme was in solution, unlike laccase adsorbed on gold, which showed only a low-potential signal. Laccase molecules were successfully imaged by AFM; obtaining a thick compact stable film on Au(111), and large aggregates forming a complex network of small branches leaving voids on the HOPG surface. Laccase-modified carbon electrodes retained significant enzymatic activity, efficiently oxidising violuric acid and reducing molecular oxygen. Explanations are proposed for how protein-film organisation affects the electrode function.

Valor pronóstico de determinados marcadores biológicos en la supervivencia global y la respuesta al tratamiento del cáncer de mama con metástasis pleurales / Prognostic value of specific biological markers in the global survival rate and response to treatment of breast cancer with pleural metastasis

Objetivo: 1. Estudiar qué tipo de marcadores biológicos tienen valor pronóstico en la supervivencia global y la respuesta al tratamiento en el cáncer de mama, a partir del descubrimiento de metástasis pleurales. 2. Examinar la influencia de la aplicación de talco intrapleural. 3. Establecer un esquema de abordaje terapéutico en base a los diferentes factores pronósticos. Pacientes y métodos: estudiamos una serie de 126 pacientes con cáncer de mama y afectación pleural metastásica. Se examinaron exhaustivamente los factores clásicos del cáncer de mama. Así mismo se calcularon el intervalo libre de enfermedad (ILE), el intervalo entre la aparición del derrame pleural y el abordaje toracoscópico del mismo, el intervalo entre la realización de la pleurodesis con talco y el exitus y el intervalo desde el diagnóstico del tumor primario hasta el exitus. Los factores biológicos (polimorfismos)fueron estudiados a partir del ADN de muestras de sangre periférica o líquido pleural crio conservado. Resultados: el derrame pleural era en el 77% de los casos la primera manifestación de recidiva de la enfermedad. El intervalo libre de enfermedad (ILE) fue de 57,9 meses (13,6-83,3). La supervivencia(expresada como mediana) desde el momento del diagnóstico del tumor primario fue de 77,5 meses (0,83-384). La media de seguimiento tras el talcaje fue de 14,6 meses. Encontramos correlación entre la edad temprana de presentación del cáncer de mama (..) (AU)

Objective: 1. To study what type of biological markers have prognostic value in the global survival rate and response to treatment of breast cancer, regarding discovery of pleural metastases. 2. To examine the influence of the application of intrapleural talc. 3. To establish a therapeutic approach outline based on the different prognosticfactors. Patients and methods: we studied a series of 126 patients with breast cancer and pleural metastasis. The classic factors of breast cancer were thoroughly examined. Also, we calculated the disease-free interval(DFI), the interval between the appearance of the pleural effusion and its thoracoscopic approach, the interval between pleurodesis with talc and death and the interval between the diagnosis of the first tumour and death. The biological factors (polymorphisms) were studied starting with the DNA of peripheral blood samples or cryopreservation of pleural liquid samples. Results: the pleural effusion was the first manifestation of a relapse of the disease in 77% of the cases. The disease-free interval (DFI) was57.9 months (13.6-83.3). The survival rate (expressed as median)from the diagnosis of the first tumour was 77.5 months (0.83-384).The average follow-up after the application of talc was 14.6 months. We found a correlation between the appearance of breast cancer(p=0.003) at a young age and the presence of the A2/A2 allele(homozygote of the CYP-17).Conclusions: the ideal profile for candidates for pleurodesis is those patients with an age >50 years, long DFI, short interval of time between the appearance of the pleural effusion and the application oftalc, absence of other metastasis at the time of thoracoscopy, positive ER (estrogen receptors) and PR (progesterone receptors) and a percentage of lymph nodes infiltrated/extracted less than 50%. The presence of mutated GSTM1 will be related with a more effective (..) (AU)

Early attack and subsequent changes produced in an industrial lignin by a fungal laccase and a laccase-mediator system: an analytical approach.

An industrial kraft pine lignin (Indulin AT, KL) was characterized and treated in both aqueous-buffered media and dioxane to water, either with a partially purified laccase from Fusarium proliferatum or with the laccase plus 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic-acid (ABTS) as mediator. The changes in the lignin after different incubation periods were analyzed through the application of high performance liquid chromatography (HPLC), UV-visible (Vis) spectroscopy and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). At the onset of incubation, laccase-treated samples showed a slight polymerization and strong modifications in UV-Vis spectra. Through Py-GC/MS, a decrease in phenolic and methoxy-bearing pyrolysis products was observed, in contrast to an increase in the more oxidized products. After longer incubation periods (48 h) a substantial polymerization was detected by HPLC, along with a decrease in the guaiacyl (G) units. In contrast, the analysis by HPLC of the samples recovered from the laccase-ABTS system (LMS) showed an intense depolymerization, accompanied by a sizeable loss in G units and a decrease in the methyl and ethyl side-chain phenolic compounds. These results provide conclusive evidence of a rapid initial attack of the industrial lignin by laccase and notable modifications in the KL after longer incubation periods with laccase or LMS.

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum.

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization-ion trap-mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60 degrees C, thermal stability for 2 h at 40 degrees C, optimum pH 3.5 (km=62 microM) measured on 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4-8 (75% stability at pH levels 2.2 and 9) for 2 h at 25 degrees C.

Tomografía computarizada abdominal

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Lignin Degradation and Modification by the Soil-Inhabiting Fungus Fusarium proliferatum.

A soil-inhabiting Fusarium proliferatum strain was capable of transforming or degrading nonlabeled and (sup14)C-labeled industrial, natural, and synthetic lignin. The mineralization rate per day (expressed as the percentage of added radioactivity recovered as to (sup14)CO(inf2)) was maximal during primary metabolism.

Short communication: Isolation of a bacterium capable of limited degradation of industrial and labelled, natural and synthetic lignins.

Pseudomonas putida, isolated from decomposing plant materials, degraded several lignin-related aromatic compounds. After 30 days of incubation in media containing polymeric Kraft-lignin (PKL), the amount of Klason lignin had decreased by about 13%. When (14)C-labelled dehydropolymers of coniferyl alcohol (DHP) lignins and (14)C-lignin-lignocelluloses were used as substrates, mineralization to (14)CO2 by the P. putida strain ranged from 1.4% to 2.1%.

Effect of Penicillium chrysogenum on Lignin Transformation.

A strain of Penicillium chrysogenum has been isolated from pine forest soils in Tenerife (Canary Islands). This strain was capable of utilizing hydroxylated and nonhydroxylated aromatic compounds, in particular cinnamic acid, as its sole carbon source. In an optimum medium with high levels of nitrogen (25.6 mM) and low levels of glucose (5.5 mM), it was able to decolorize Poly B-411 and to transform kraft, organosolv, and synthetic dehydrogenative polymerisate lignins. After 30 days of incubation, the amount of recovered kraft lignin was reduced to 83.5 and 91.3% of that estimated for uninoculated controls by spectrophotometry and klason lignin, respectively. At the same time, the pattern of molecular mass distribution of the lignin remaining in cultures was changed. The amount of organosolv lignin recovered from cultures was reduced to 90.1 and 94.6% of the initial amount as evaluated by spectrophotometry and klason lignin, respectively. About 6% of total applied radioactivity of OCH(3)-organosolv lignin was recovered as CO(2) after 30 days of incubation, and 18.5% of radioactivity from insoluble OCH(3)-organosolv lignin was solubilized. After 26 days of incubation, 2.9% of C-beta-dehydrogenative polymerisate and 4.1% of C-ring-dehydrogenative polymerisate evolved as CO(2).

Oxidation of aromatic aldehydes by Serratia marcescens.

Cell suspensions of Serratia marcescens catalyzed the oxidation of aromatic aldehydes into the corresponding acids in high yield under mild conditions.

A protease with staphylolytic activity from Pseudomonas aeruginosa PAKS I.

The supernatant from broth cultures of Pseudomonas aeruginosa PAKS I contains two different enzymes with staphylolytic activity. One of them, namely staphylolytic enzyme, seems to be specific for glycine-rich cross-links present in the cell wall of different Gram-positive bacteria and has been previously characterized. In addition to the staphylolytic activity, the second protein which we propose to be a staphylolytic protease, has proteolytic activity against casein. This enzyme is approximately 33 kDa, has an isoelectric point ranging from 7.3 to 8.1 and an optimum pH value of 8.0 for casein hydrolysis. Staphylolytic protease was detected in the extracellular medium after 12 h of cell growth. Immunocytochemical studies suggest that the protease is located within the periplasmic space of P. aeruginosa.

Staphylolytic enzyme from Pseudomonas aeruginosa: characterization and immunocytochemical localization.

Staphylolytic enzyme, a specific peptidase produced by Pseudomonas aeruginosa, has been characterized by using immunochemical procedures. Lytic activity was detected in the extracellular medium of Pseudomonas cultures at the beginning of the stationary growth phase. No activity was detected in bacterial cells. However, lytic protein antigen was present in periplasmic and cytoplasmic fractions, suggesting that staphylolytic enzyme is synthesized as an inactive precursor which becomes active during translocation to the extracellular broth. Results obtained in immunolocalization experiments indicate the presence of the precursor in the outer part of cells. The export pathway of staphylolytic enzyme through the periplasmic space is proposed.

L-form-like colonies of Staphylococcus aureus induced by an extracellular lytic enzyme from Pseudomonas aeruginosa.

An extracellular enzyme produced by Pseudomonas aeruginosa had a lytic effect on lyophilized Staphylococcus aureus cells. It was purified from the culture supernatant by ammonium sulfate fractionation followed by column chromatography with P cellulose and Sephadex G-50. The molecular weight of the enzyme was estimated to be 19,000 +/- 1,750 with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI of the enzyme was estimated to be 8.5 with isoelectric focusing. The enzyme was inactive in 4% NaC1-40 mM sodium phosphate buffer or at pH values lower than 6.0 or higher than 11.0; however, it was not affected by 1 M sucrose or 0.25% heat-denatured horse serum. The action of the enzyme on cultures of S. aureus resulted in the presence of many cells lacking cell walls. In addition, when cultivation was carried out on osmotically stabilized solid media, these cell wall-deficient cell developed in L-form colonies.

Purification and peptidase activity of a bacteriolytic extracellular enzyme from Pseudomonas aeruginosa.

A bacteriolytic enzyme excreted by Pseudomonas aeruginosa Paks I was purified: samples were found to be homogeneous by gel filtration chromatography, ion exchange chromatography using CM-cellulose, immunoelectrophoresis, PAGE and SDS-PAGE. The molecular weight of the lytic enzyme was estimated to be 15,000-19,000. The enzyme was active on Gram-positive bacteria with glycine-containing interpeptide bridges in their murein layers. In addition, this lytic enzyme showed peptidase activity catalysing the hydrolysis of pentaglycine peptides into tri- and diglycine peptides.

Effects of staphylolytic enzymes from Pseudomonas aeruginosa on the growth and ultrastructure of Staphylococcus aureus.

Pseudomonas aeruginosa produces two extracellular staphylolytic enzymes able to lyse Staphylococcus aureus cells when they are added to liquid cultures of S. aureus. In addition, when cultivation was carried out in the presence of both lytic enzymes and 1 M sucrose, the staphylococci either lacked cell walls or showed damaged walls. Lytic activity-resistant cells of S. aureus were also detected.