Anna Matuszewska

Fungal laccase: properties and activity on lignin

The sources of ligninocellulose that occur in various forms in nature are so vast that they can only be compared to those of water. The results of several, more recent experiments showed that laccase probably possesses the big ability for “lignin‐barrier” breakdown of ligninocellulose. The degradation of this compound is currently understood as an enzymatic process mediated by small molecules, therefore, this review will focus on the role of these mediators and radicals working in concert with enzymes. The fungi having a versatile machinery of enzymes are able to attack directly the “lignin‐barrier” or can use a multienzyme system including “feed‐back” type enzymes allowing for simultaneous transformation of lignin and carbohydrate compounds.

New Bioactive Fungal Molecules with High Antioxidant and Antimicrobial Capacity Isolated from Cerrena unicolor Idiophasic Cultures

Three bioactive fractions, extracellular laccase (ex-LAC), crude endopolysaccharides (c-EPL), and a low molecular subfraction of secondary metabolites (ex-LMS), were isolated from the idiophasic cultures of the white rot fungus Cerrena unicolor. For the first time, we determined the antioxidant properties of these samples by chemiluminometric measurement (a) and assessment of the scavenging effect on ABTS (b) and the DPPH reduction rate (c). The highest reducing capability was found for the ex-LMS fraction: 39–90% for (a), 20–90% for (b), and 10–59% for (c) at the concentration of 6.25–800 µg/mL. The scavenging abilities of the C. unicolor c-EPL were between 36 and 70% for (a), 2 and 60% for (b), and 28 and 32% for (c) at the concentration of 6.25–800 µg/mL. A very high prooxidative potential was observed for the ex-LAC probes. The preliminary toxicity tests were done using the Microtox system and revealed the following percentage of the toxic effect against Vibrio fischeri: 85.37% for c-EPL, 50.67% for ex-LAC, and 99.8% for ex-LMS, respectively. The ex-LAC sample showed the antibacterial activity against Escherichia coli, c-EPL against Staphylococcus aureus, and ex-LMS against both bacterial strains, respectively, but the stronger inhibitory effect was exerted on S. aureus.

Fungus Cerrena unicolor as an effective source of new antiviral, immunomodulatory, and anticancer compounds

In the report, three bioactive fractions from Cerrena unicolor: laccase (LAC), endopolysaccharides (c-EPL), and low molecular weight (ex-LMS) were tested for the first time towards their antiviral, immunostimulatory, cytotoxic and antiproliferative effect. The immunomodulatory activity was studied by means of THP-1-derived macrophages able to synthesize and secrete IL-6 and TNF-α. We used cervical carcinoma cell lines SiHa (ATCC, HTB-35) and CaSki (ATCC, CRL 1550) to determine antitumor activity and human skin fibroblasts (HSF) as a control. SiHa and L929 cell lines were used in the antiviral activity assay to propagate HHV-1 and EMCV, respectively. LAC was the most active against HSV at an early stage of viral replication, whereas the activity of laccase against EMCV was evident after incubation of the virus with LAC before and after the adsorption step. Moreover, the investigations showed that the fungal c-EPL fraction stimulated the production and secretion of TNF-α and IL-6 by THP-1-derived macrophages up to a level of 2000 pg/ml and 400 pg/ml, respectively. It was indicated for the first time that the LAC and ex-LMS fractions exhibited anticancer activity. This resulted from their cytotoxic or antiproliferative action against the investigated tumor cells at concentrations above 250 μg/ml and 10 μg/ml, respectively.

Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells.

Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms.

Fungal polysaccharides as a water-adsorbing material in esters production with the use of lipase from Rhizomucor variabilis

The extracellular crude Rhizomucor variabilis lipase was used for synthesis of flavor ester butyl caprylate and 1-butyl oleate often used as a diesel additive, a polyvinyl chloride plasticizer, a water-resisting agent, and an additive to hydraulic fluids. The influence of various reaction parameters such as the molar ratio, time, enzyme and substrate concentration, and effect of various fungal polysaccharides was estimated. The rate of catalyzed synthesis of esters largely depends on the solvent medium, and the maximum activity was found when n-hexane was used as a solvent. The maximum conversion yield of 58.2% and 59.3% was obtained for butyl caprylate and butyl oleate, respectively, under the following conditions: amount of free lipase 500 U; caprylic acid:butanol molar ratio 1:1; oleic acid:butanol molar ratio 2:1. The addition of naturally obtained fungal polysaccharides significantly enhanced the ester synthesis. The highest conversion rate of 95.2% was observed for butyl caprylate in the presence of AbEPS after 24 h with 500 U of free R. variabilis lipase. In the case of butyl oleate synthesis in the presence of LsPS, a maximum conversion yield of 91.2% was observed after the 24-h reaction.

Antimelanomic Effects of High- and Low-Molecular Weight Bioactive Subfractions Isolated from the Mossy Maze Mushroom, Cerrena unicolor (Agaricomycetes)

Three bioactive fractions isolated from Cerrena unicolor cultures-crude endopolysaccharide (c-EPS), laccase, and a subfraction of low-molecular weight secondary metabolites-were used to determine potential cytotoxic effects on the mouse melanoma B16-F10 cell line (American Type Culture Collection CRL-6475). The results obtained prove that all examined fractions exhibited activity against the investigated tumor cells. In addition, an evident immunomodulatory effect of the c-EPS fraction was observed. Our results show that the levels of 2 cytokines (tumor necrosis factor-a and chemokine ligand 2) in mouse inner medullary collecting duct mIMCD-3 cells (American Type Culture Collection CRL-2123) stimulated by c-EPS were significantly higher. A lipopolysaccharide model was used at the same concentration (10 μg/mL) as a positive control.

Determination of pamidronate in bisphosphonate-enriched bone cement by ion-pair hplc and capillary electrophoresis

Abstract The presence of pamidronate during local use of bisphosphonates (BP)-enriched bone cement was determined. The question was whether pamidronate implanted into the bone cement is eluted. The study was performed on 10 probes of BP-enriched bone cement located in 0.9% NaCl. The probes were incubated for 3 and 6 weeks. Ion-pair HPLC was used for the detection of pamidronate. Then, capillary electrophoresis was applied for quantitative analysis of pamidronate in the 3rd and 6th week after incubation. The presence of pamidronate, eluted from BP-enriched bone cement into 0.9% NaCl solution 3 and 6 weeks after incubation, was demonstrated. These results may explain the changes in the level of cytokine RANKL and bone turnover marker osteoprotegrin in rats’ serum treated with BP-enriched bone cement 3 and 6 weeks after surgery. The possibility of effective local use of BP-enriched bone cement in veterinary medicine was underlined. The results, and the former conducted research, point out that the clinical applications of BP-enriched bone cement in vivo may have some validity in the future.

Anticancer, antioxidant, and antibacterial activities of low molecular weight bioactive subfractions isolated from cultures of wood degrading fungus Cerrena unicolor

The aim of this study is to investigate in vitro the anticancer, antioxidant, and antibacterial activities of three low molecular weight subfractions I, II and III isolated from secondary metabolites produced by the wood degrading fungus Cerrena unicolor. The present study demonstrated that the low molecular weight subfractions III exhibited the strongest inhibitory activity towards breast carcinoma cells MDA-MB-231, prostatic carcinoma cells PC3, and breast cancer cells MCF7 with the half-maximal inhibitory concentration (IC50) value of 52,25 μg/mL, 60,66 μg/mL, and 54,92 μg/mL, respectively. The highest percentage of inhibition was noted at a concentration of 300 μg/mL in all the examined tumor lines. A significant percentage (59.08%) of ex-LMSIII inhibition of the MDA-MB-231 tumor line was reached at a concentration of 15 μg/ml, while the concentration applied did not affect normal human fibroblast cells. The low molecular weight subfraction III was the most effective and additionally showed the highest free radical 1,1-diphenyl-2-picryl-hydrazyl scavenging activity (IC50 20.39 μg/mL) followed by the low molecular weight subfraction I (IC50 64.14 μg/mL) and II (IC50 49.22 μg/mL). The antibacterial activity of the tested preparations was evaluated against three microorganisms: Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The MIC minimal inhibitory concentration (MIC) values for the low molecular weight subfraction I, II, and III showed a stronger inhibition effect on S. aureus than on B. subtilis and E. coli cells. The MIC values for the low molecular weight subfraction II against S. aureus, B. subtilis, and E. coli were 6.25, 12.5, and 100 mg/mL, respectively.

The presence of pamidronate in bone cement affects serum biochemical markers in the rat

Abstract The main aim of the study was to assess whether the presence of biphosphate pamidronate (PA) in the cement implanted into the tibial bones had any effect on the chosen biochemical markers in rat’s serum characterising homeostasis. Forty adult male Wistar rats were divided into two control groups and two experimental groups. Tibial bone of rats in the experimental groups was implanted with PA-enriched cement, whereas the bone in control-group’s rats was implanted with cement without PA. Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) were determined three and six weeks after the surgery. Statistically significant differences in the activities of AST and CK of the rats after implantation with non-enriched cement when compared to rats given PA-enriched cement implantation, were found. Six weeks after treatment, AST levels decreased significantly in rats with PA-enriched cement, whereas rats in the control group (implanted with non-enriched cement) demonstrated a significant increase in AST activity in comparison to the same values determined after three weeks and values of PA-enriched cement rats determined after six weeks. The activities of CK were higher in rats with PA-enriched implants than in the control group three weeks after surgery, but six weeks after the treatment, rats implanted with enriched cement reached lower values than animals implanted with non-enriched cement. The use of PA in the cement had also some positive effect on the homeostasis of the rats after the surgery and a positive influence on the post operative muscle regeneration process.