Krzysztof Grzywnowicz

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.

The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

AimsThe aim of this study was investigation of the response of R. leguminosarum bv. trifolii wild-type and its two rosR and pssA mutant strains impaired in exopolysaccharide (EPS) synthesis to oxidative stress conditions caused by two prooxidants: a superoxide anion generator- menadione (MQ) and hydrogen peroxide (H2O2).MethodsThe levels of enzymatic (catalase, superoxide dismutase, pectinase and β-glucosidase) and non-enzymatic (superoxide anion generator, formaldehyde, phenolic compounds) biomarkers were monitored using biochemical methods in both the supernatants and rhizobial cells after treatment with 0.3 mM MQ and 1.5 mM H2O2. The viability of bacterial cells was estimated using fluorescent dyes and confocal laser scanning microscopy. In addition, the effect of prooxidants on symbiosis of the R. leguminosarum bv. trifolii strains with clover was established.ResultsThe tested stress factors significantly changed enzymatic patterns of the rhizobial strains, and the wild-type strain proved to be more resistant to these prooxidants than both pssA and rosR mutants. Significantly higher activities of both catalase and superoxide dismutase have been detected in these mutants in comparison to the wild-type strain. H2O2 and MQ also increased the levels of pectinase and β-glucosidase activities in the tested strains. Moreover, pre-incubation of R. leguminosarum bv. trifolii strains with the prooxidants negatively affected the viability of bacterial cells and the number of nodules elicited on clover plants.ConclusionsEPS produced in large amounts by R. leguminosarum bv. trifolii plays a significant protective role as a barrier against oxidative stress factors and during symbiotic interactions with clover plants.

Profiles of the body-surface proteolytic system of honey bee queens, workers and drones: Ontogenetic and seasonal changes in proteases and their natural inhibitors*

We describe how protease and protease inhibitor activity patterns vary during ontogenesis, with season, and in relation to caste and sex in the honey bee (Apis mellifera). Extraction of body surfaces with water and detergent was followed by the in vitro analysis of proteolytic activity and protease inhibitor level, as well as the electrophoretic separation of extracts in polyacrylamide gels. In in vitro experiments, we compared two groups of detectable proteolytic activities: neutral and alkaline water-soluble versus acidic detergent-soluble. The most active proteases appeared to be acidic ones and were detected on drone pupae in spring. The most distinct and most active protease bands in electrophoretic separations were those obtained for neutral and alkaline activities on queens in all seasons. The highest levels of protease inhibitor activities in vitro were obtained from worker samples in all seasons. The enzymatic properties suggest that all catalytic types of proteases were present in the extracts, but at different activity levels, depending on pH: asparagine and cysteine proteases at pH 2.5, cysteine proteases and metalloproteases at pH 7.0, and serine proteases at pH 11.5, respectively.ZusammenfassungBei verschiedenen Organismen, einschliesslich Schaben, Amphibien und Menschen, wurde bereits eine beschränkte Anzahl an Proteasen und/oder Proteaseinhibitoren auf der Körperoberfläche identifiziert. Dabei sind die Unterschiede innerhalb der Invertebraten und im Vergleich mit Vertebraten nur relativ gering. Untersuchungen basierend auf dem Genom der Honigbienen weisen bereits auf eine bestimmte Anzahl neuer Proteine, einschliesslich Kutikulaproteinen, hin, die auch Proteasen umfassen könnten. Ziel dieser Studie war die Beschreibung der Muster, der in vitro Aktivität und der Zymogramme der Proteasen der Körperoberfläche und ihrer natürlichen Inhibitoren im Verlauf der Ontogenese der Honigbiene (Apis mellifera). Dabei wurden auch Kasten- und Saisonunterschiede in Betracht gezogen.Adulte Honigbienen wurden entweder am Stockeingang abgefangen (Arbeiterinnen) oder aus dem Volk entommen (Königinnen und Drohnen). Puppen, Larven und Eier wurden aus Brutwaben gewonnen. Larven der ersten drei Larvenstadien wurden als „kleine Larven“ und die des vierten und fünften Larvenstadiums als „grosse Larven“ gepoolt. Alle Proben wurden mit destilliertem Wasser abgewaschen, um Verunreinigungen und nicht festhaftende Mikroorganismen zu entfernen. Anschliessend wurde mittels feuchter Baumwollstäbchen die Körperoberfläche abgewaschen. Aus den Baumwollstäbchen wurden dann die biologischen Proben ausgepresst. Für eine zweite Waschung wurde destilliertes Wasser mit 1 % Detergenz verwendet. Allgemeine Proteasenaktivitäten wurden am optimalen pH mittels Hämoglobin als Substrat bestimmt (Tab. I) In den Extrakten waren alle katalytischen Typen an Proteasen vertreten. Bei pH 2,5 hatten die Asparaginproteasen (inhibiert durch Pepstatin und DAN) und Cysteinproteasen (inhibiert durch Iodoacetamid und pCMB) ihre höchsten Werte; bei pH 7,0 erreichten die Cysteinproteasen und Metalloproteasen (inhibiert durch o-Phenantrolin) und bei pH 11,5 die Serinproteasen (inhibiert durch PMSF und STI) die höchsten Aktivitätswerte.Die Bestimmung der allgemeinen Niveaus der Aktivitäten der Proteaseninhibitoren wurde mit Pepsin als Proteasemarker bei saurem pH und mit Trypsin als Markerprotease für neutrale und basische pH-Werte durchgeführt. Elektrophoresen in 10 % Gelen erfolgten bei nichtdenaturierenden Bedingungen, mit 1 % Gelatine als vorgegebenem Proteinsubstrat. In in vitro-Versuchen verglichen wir unter identischen Bedingungen zwei Gruppen proteolytischer Aktivitäten: die neutralen und basischen wasserlöslichen und die sauren, detergenzlöslichen. Die aktivsten Proteasen waren saure Proteasen auf Drohnenpuppen, die im Frühling gesammelt worden waren (Abb. 2), neutrale und basische Proteasen auf adulten Drohnen im Sommer (Abb. 3), neutrale auf adulten Königinnen im Herbst, sowie alle Proteasentypen auf Königinnenpuppen im Frühjahr (Abb. 4 und Abb. 2). Die geringsten Proteaseaktivitäten fanden wir auf Arbeiterinnen im Frühjar (Abb. 2) und auf Drohnen im Herbst (Abb. 4). In allen Fällen waren die Aktivitäten auf Puppen und Imagines höher als auf den anderen Stadien. Im zymographischen Vergleich in Gelen mit Gelatine zeigten sich einige Unterschiede. Die schärfsten Banden mit der höchsten Aktivität fanden wir für neutrale und basische Werte auf Königinnen in allen Jahreszeiten. Die verschwommensten Banden waren für neutrale und basische Werte auf Drohnen in allen Jahreszeiten zu verzeichnen. Bei Arbeiterinnenproben erhielten wir für alle Jahreszeiten sowohl klare als auch verschwommen Banden. Letzteres steht in Übereinstimmung mit den niedrigen Proteaseaktivitäten, die wir für Arbeiterinnen in den in vitro-Versuchen gefunden hatten. Vergleichbar hohe Werte für Proteaseinhibitoren fanden wir bei Drohnen im Sommer (Abb. 6) und bei adulten Königinnen im Herbst (Abb. 7).

Partial Purification of Proteinase K Inhibitors from Liquid-Cultured Mycelia of the White Rot Basidiomycete Trametes versicolor

Novel protease inhibitors were isolated from liquid-cultured mycelia of the white rot fungus Trametes versicolor. Two bands of antiproteinase K activity, TvPI-A and TvPI-B, were detected in the crude cell extract by native polyacrylamide gel electrophoresis (PAGE). Proteins corresponding to TvPI-A were purified by heat treatment, anion-exchange chromatography, and gel filtration. Sodium dodecyl sulfate (SDS)-PAGE demonstrated the presence of three proteins with molecular masses of 14.5, 16.6, and 20 kDa, respectively. T. versicolor protease inhibitors suppressed the activity of proteinase K and, to a smaller extent, of Carlsberg subtilisin, whereas trypsin and chymotrypsins were not inhibited. The inhibitors were acidic proteins and showed remarkable heat stability. To our knowledge, this is the first report about proteinase K inhibitors from fungi.

Anti-Candida albicans action of the glyco-protein complex purified from metabolites of gut bacterium Raoultella ornithinolytica isolated from earthworms Dendrobaena veneta

The aim of our research was to isolate the compounds from the metabolites of Raoultella ornithinolytica with the activity against Candida albicans and to analyse the action of the compounds on the metabolic activity and morphology of the fungus cells.

Antitumour and apoptotic effects of a novel Tris‐peptide complex obtained after isolation of Raoultella ornithinolytica extracellular metabolites

The characterization of the antitumour activity and chemical identification of the compounds obtained after the isolation of extracellular metabolites of bacteria Raoultella ornithinolytica.

Antimycobacterial action of a new glycolipid-peptide complex obtained from extracellular metabolites of Raoultella ornithinolytica

In this paper, an antimycobacterial component of extracellular metabolites of a gut bacterium Raoultella ornithinolytica from D. veneta earthworms was isolated and its antimycobacterial action was tested using Mycobacterium smegmatis. After incubation with the complex obtained, formation of pores and furrows in cell walls was observed using microscopic techniques. The cells lost their shape, stuck together and formed clusters. Surface‐enhanced Raman spectroscopy analysis showed that, after incubation, the complex was attached to the cell walls of the Mycobacterium. Analyses of the component performed with Fourier transform infrared spectroscopy demonstrated high similarity to a bacteriocin nisin, but energy dispersive X‐ray spectroscopy analysis revealed differences in the elemental composition of this antimicrobial peptide. The component with antimycobacterial activity was identified using mass spectrometry techniques as a glycolipid–peptide complex. As it exhibits no cytotoxicity on normal human fibroblasts, the glycolipid–peptide complex appears to be a promising compound for investigations of its activity against pathogenic mycobacteria.

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.