Jerzy Wydrych

Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme.

The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.

Exopolysaccharide from Ganoderma applanatum as a Promising Bioactive Compound with Cytostatic and Antibacterial Properties

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85u2009µg/mL and 228.5u2009µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17u2009pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24u2009h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

Can 2-oxoglutarate prevent changes in bone evoked by omeprazole?

OBJECTIVEnProton-pump inhibitors, such as omeprazole, are widely used in the prevention and treatment of gastroesophageal diseases. However, an association between proton-pump inhibitors and the increased risk of bone fractures has been observed, especially in patients treated for extended periods. Conversely, 2-oxoglutarate, a precursor of hydroxyproline, the most abundant amino acid in bone collagen, counteracts the bone loss. The aim of the present study was to elucidate the influence of omeprazole on bone and investigate whether dietary 2-oxoglutarate supplementation could prevent the effects of omeprazole.nnnMETHODSnEighteen male Sprague-Dawley rats were used. Rats received omeprazole in the diet and 2-oxoglutarate in the drinking water. Body and organ weights and serum concentrations of cholecystokinin and gastrin were measured. The femurs, tibias, and calvarias were collected. Histomorphometric analysis of bone and cartilage tissues was conducted. Bone densitometric and peripheral quantitative computed tomographic analyses of the femur and tibia were performed.nnnRESULTSnOmeprazole decreased the femur and tibia weights, the mechanical properties of the femur, the volumetric bone density and content, the trabecular and cortical bone mineral content, the total, trabecular, and cortical bone areas, the mean cortical thickness, and the periosteal circumference of the femur. Omeprazole had a minor effect on the examined bone morphology and exerted negligible effects on the cartilage. 2-Oxoglutarate lowered the gastrin concentration.nnnCONCLUSIONSnOmeprazole treatment exerts its effects mostly on bone mineralization and cancellous bone, adversely affecting bone properties. This adverse effect of omeprazole was not markedly abolished by 2-oxoglutaric acid, which acted as an anti-hypergastrinemic agent.

Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells

The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.

Studies on localization and protein ligands of Galleria mellonella apolipophorin III during immune response against different pathogens

A lipid-binding protein apolipophorin III (apoLp-III), an exchangeable component of lipophorin particles, is involved in lipid transport and immune response in insects. In Galleria mellonella, apoLp-III binding to high-density lipophorins and formation of low-density lipophorin complexes upon immune challenge was reported. However, an unanswered question remains whether apoLp-III could form different complexes in a pathogen-dependent manner. Here we report on pathogen- and time-dependent alterations in the level of apoLp-III free and lipophorin-bound form that occur in the hemolymph and hemocytes shortly after immunization of G. mellonella larvae with different pathogens, i.e. Gram-negative bacterium Escherichia coli, Gram-positive bacterium Micrococcus luteus, yeast-like fungus Candida albicans, and filamentous fungus Fusarium oxysporum. These changes were accompanied by differently persistent re-localization of apoLp-III in the hemocytes. The apoLp-III-interacting proteins were recovered from immune hemolymph by affinity chromatography on a Sepharose bed with immobilized anti-apoLp-III antibodies. ApoLp-I, apoLp-II, hexamerin, and arylphorin were identified as main components that bound to apoLp-III; the N-terminal amino acid sequences of G. mellonella apoLp-I and apoLp-II were determined for the first time. In the recovered complexes, the pathogen-dependent differences in the content of individual apolipophorins were detected. Apolipophorins may thus be postulated as signaling molecules responding in an immunogen-dependent manner in early steps of G. mellonella immune response.

Bacterial exopolysaccharides as a modern biotechnological tool for modification of fungal laccase properties and metal ion binding

Four bacterial EPSs extracted from Rhizobium leguminosarum bv. trifolii Rt24.2, Sinorhizobium meliloti Rm1021, Bradyrhizobium japonicum USDA110, and Bradyrhizobium elkanii USDA76 were determined towards their metal ion adsorption properties and possible modification of Cerrena unicolor laccase properties. The highest magnesium and iron ion-sorption capacity (~u200942 and ~u200914.5%, respectively) was observed for EPS isolated from B. japonicum USDA110. An evident influence of EPSs on the stability of laccase compared to the control values (without EPSs) was shown after 30-day incubation at 25xa0°C. The residual activity of laccases was obtained in the presence of Rh76EPS and Rh1021EPS, i.e., 49.5 and 41.5% of the initial catalytic activity, respectively. This result was confirmed by native PAGE electrophoresis. The EPS effect on laccase stability at different pH (from 3.8 to 7.0) was also estimated. The most significant changes at the optimum pH value (pH 5.8) was observed in samples of laccase stabilized by Rh76EPS and Rh1021EPS. Cyclic voltamperometry was used for analysis of electrochemical parameters of laccase stabilized by bacterial EPS and immobilized on single-walled carbon nanotubes (SWCNTs) with aryl residues. Laccases with Rh76EPS and Rh1021EPS had an evident shift of the value of the redox potential compared to the control without EPS addition. In conclusion, the results obtained in this work present a new potential use of bacterial EPSs as a metal-binding component and a modulator of laccase properties especially stability of enzyme activity, which can be a very effective tool in biotechnology and industrial applications.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

Age- and Glucocorticoid-Dependent Leptin Release by Horse Adipose Tissue: In Vitro Study

ABSTRACT In mammals, corticosteroids overall play the greatest role in stimulation of leptin synthesis and release from the adipose tissue. Some studies have shown that changes in plasma leptin and cortisol concentrations in horses can occur independently of each other. The aim of our study on horses was to evaluate the effect of different ages on the glucocorticoid‐dependent leptin release from isolated adipose tissue samples in primary culture. Subcutaneous adipose tissue samples were collected from 25 cold‐blood horses aged 7 months to 11 years. Samples of adipose tissue were incubated (200 mg/mL) under control conditions (Dx0), or treated with one of two doses of dexamethasone (100 ng/mL—Dx100 and 1,000 ng/mL—Dx1000) for 12 hours. Histological examination was also conducted to obtain the mean number of adipocytes in the field of view. The medium leptin concentration was analyzed in relation to the number of adipocytes in the tissue samples. A significantly higher medium leptin concentration in Dx1000 than in Dx0 was observed only in tissue samples from foals (7–12 months of age, n = 6). Significant negative correlations were found between the age of horses and the difference in medium leptin levels Dx1000 − Dx0, as well as (Dx1000 − Dx0)/million adipocytes, and (Dx1000 − Dx100)/million adipocytes. The corticosteroids‐induced secretion of leptin decreased with the age of horses. HIGHLIGHTSThe effect of horses age on the glucocorticoid‐dependent leptin release was studied.Adipose tissue samples taken from horses of different age were studied in vitro.Age of horses correlated negatively with the dexamethasone‐induced leptin release.The corticosteroids‐induced leptin release decreases with the age of horses.

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.