Induction and Characterization of Laccase in the Ligninolytic Fungus Pleurotus eryngii

Two protein bands with laccase activity were found after PAGE of culture liquid or mycelium extract of Pleurotus eryngii, grown on glucose–ammonium tartrate–yeast extract medium with and without inducers. A major and a minor laccase band were observed in the basal medium. The intensity of the major band (laccase I) did not change after the addition of inducers. However, the minor band (laccase II), characterized by higher electrophoretic mobility, was strongly induced by wheat–straw alkalilignin and vanillic and veratric acids. Laccase activity in the basal medium had an optimum pH of 4.5 and was stable from pH 3 to 10 during 24 h at room temperature. This enzyme had wide substrate specificity on hydroquinones, methoxy-substituted monophenols, and aromatic amines. In general, laccase activity was found only with compounds having a redox potential lower than 0.5 mV. The highest activity was obtained with methoxy- and methyl-substituted p-hydroquinones and aromatic diamines. Some activity also occurred with the aliphatic compound 3,5-cyclohexadiene-1,2-diol. Received: 22 April 1996 / Accepted: 29 June 1996     

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Effect of organic and conventional farming on soil bacterial diversity of pecan tree (Carya illinoensis K. Kosh) orchard across two phenological stages

We described the bacterial diversity of walnut grove soils under organic and conventional farming. The bacterial communities of rhizospheric and nonrhizospheric soils of pecan tree (Carya illinoensis K. Koch) were compared considering two phenological stages (sprouting and ripening). Sixteen operational taxonomic units (OTUs) were identified significantly more abundant according to the plant development, only one according to the farming condition, and none according to the soil origin. The OTUs specificaly abundant according to plant development included Actinobateria (2) and Betaproteobacteria (1) related OTUs more abundant at the sprouting stage, while at the fruit ripening (FR) stage the more abundant OTUs were related to Actinobacteria (6), Alphaproteobacteria (6), and unclassified Bacteria (1). The Gaiellaceae OTU18 (Actinobacteria) was more abundant under conventional farming. Thus, our study revealed that the plant development stage was the main factor shaping the bacterial community structure, while less influence was noticed for the farming condition. The bacterial communities exhibited specific metabolic capacities, a large range of carbon sources being used at the FR stage. The identified OTUs specifically more abundant represent indicators providing useful information on soil condition, potential tools for the management of soil bacterial communities.     

Radiation techniques used in patients with breast cancer: Results of a survey in Spain

AimTo evaluate the resources and techniques used in the irradiation of patients with breast cancer after lumpectomy or mastectomy and the status of implementation of new techniques and therapeutic schedules in our country.BackgroundThe demand for cancer care has increased among the Spanish population, as long as cancer treatment innovations have proliferated. Radiation therapy in breast cancer has evolved exponentially in recent years with the implementation of three-dimensional conformal radiotherapy, intensity modulated radiotherapy, image guided radiotherapy and hypofractionation.Material and MethodsAn original survey questionnaire was sent to institutions participating in the SEOR-Mama group (GEORM). In total, the standards of practice in 969 patients with breast cancer after surgery were evaluated.ResultsThe response rate was 70% (28/40 centers). In 98.5% of cases 3D conformal treatment was used. All the institutions employed CT-based planning treatment. Boost was performed in 56.4% of patients: electrons in 59.8%, photons in 23.7% and HDR brachytherapy in 8.8%. Fractionation was standard in 93.1% of patients. Supine position was the most frequent. Only 3 centers used prone position. The common organs of risk delimited were: homolateral lung (80.8%) and heart (80.8%). In 84% histograms were used. An 80.8% of the centers used isocentric technique. In 62.5% asymmetric fields were employed. CTV was delimited in 46.2%, PTV in 65% and both in 38.5%. A 65% of the centers checked with portal films. IMRT and hypofractionation were used in 1% and in 5.5% respectively.ConclusionIn most of centers, 3D conformal treatment and CT-based planning treatment were used. IMRT and hypofractionation are currently poorly implemented in Spain.     

Effect of mixing on biogas production during mesophilic anaerobic digestion of screened dairy manure in a pilot plant

The effect of mixing on biogas production of a 1.5‐m³ pilot continuous stirred tank reactor (CSTR) processing screened dairy manure was evaluated. Mixing was carried out by recirculation of reactor content with a mono pump. The experiment was conducted at a controlled temperature of 37±1°C and hydraulic retention times (HRTs) of 20 and 10 days. The effect of continuous and intermittent operation of the recirculation pump on biogas production was studied. At 10 days of HRT, the results showed a minimal influence of recirculation rate on biogas production and that continuous recirculation did not improve reactor performance. At 20 days of HRT, the recirculation rate did not affect reactor performance. Combination of low solid content in feed animal slurry and long HRTs results in minimal mixing requirements for anaerobic digestion.     

Combinatorial gene therapy renders increased survival in cirrhotic rats

Background  

Liver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by acummulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals.     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.     

Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.