Modified polymeric biomaterials with antimicrobial and immunomodulating properties.

The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.

Investigation of the Membrane Localization and Interaction of Selected Flavonoids by NMR and FTIR Spectroscopy.

In this report, we discuss the effects of undescribed flavone derivatives, HZ4 and SP9, newly isolated from the aerial parts of Hottonia palustris L. and Scleranthus perennis L. on membranes. Interaction of flavonoids with lipid bilayers is important for medicinal applications. The experiments were performed with FTIR and NMR techniques on liposomes prepared from DPPC (dipalmitoylphosphatidylcholine) and EYPC (egg yolk phosphatidylcholine). The data showed that the examined polyphenols incorporate into the polar head group region of DPPC phospholipids at both 25 °C and 45 °C. At the lower temperature, a slight effect in the spectral region of the ester carbonyl group is observed. In contrast, at 45 °C, both compounds bring about the changes in the spectral regions attributed to antisymmetric and symmetric stretching vibrations of CH2 and CH3 moieties. Similarly, as in DPPC lipids, the tested compounds interact with the fingerprint region of the polar head groups of the EYPC lipids and cause its reorganization. The outcomes obtained by NMR analyses confirmed the localization of both flavonoids in the polar heads zone. Unraveled effects of HZ4 and SP9 in respect to lipid bilayers can partly determine their biological activities and are crucial for their usability in medicine as disease-preventing phytochemicals.

Polar localization of new flavonoids from aerial parts of Scleranthus perennis and Hottonia palustris and their modulatory action on lipid membranes properties.

The aim of this study was to characterize, for the first time, the interactions, location, and influence of flavonoids isolated from aerial parts of Scleranthus perennis (Caryophyllaceae) and Hottonia palustris (Primulaceae) on the properties of model lipid membranes prepared from dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine (EYPC). The tested compounds incorporated into liposomes into the region of the polar heads or at the water/membrane interface of DPPC phospholipids. Spectral effects accompanying the presence of polyphenols revealed their effect on ester carbonyl groups apart from SP8. All polyphenols brought about reorganization of the polar zone of liposomes as it was observed by FTIR technique. Additionally, fluidization effect was noted in the region of symmetric and antisymmetric stretching vibrations of the CH2 and CH3 groups with exception to HZ2 and HZ3. Similarly, in EYPC liposomes, they interacted mainly with the regions of the choline heads of the lipids and had various effects on the carbonyl ester groups with exception to SP8. The region of polar head groups is restructured due to the presence of the additives in liposomes. The outcomes obtained using the NMR technique confirmed the locations of all of the tested compounds in the polar zone and indicated a flavonoid-dependent modifying effect towards lipid membranes. HZ1 and SP8 raised motional freedom in this region whereas opposite effect was revealed for HZ2 and HZ3. In the hydrophobic region restricted mobility was noted. In this report we discuss the mechanism of previously undescribed flavonoids in terms of their actions on membranes.

Lensoside Aß as an Adjuvant to the Anti-Glioma Potential of Sorafenib.

AIM: The anti-glioma effect of lensoside Aß alone and in combination with sorafenib (pro-survival Raf kinase inhibitor) was evaluated for the first time in terms of programmed cell death induction in anaplastic astrocytoma and glioblastoma multiforme cell lines as an experimental model. Apoptosis, autophagy, and necrosis were identified microscopically (fluorescence and scanning microscopes) and confirmed by flow cytometry (mitochondrial membrane potential MMP and cell death). The expression of apoptotic (caspase 3) and autophagic markers (beclin 1) as well as Raf kinase were estimated by immunoblotting. The FTIR method was used to determine the interaction of the studied drugs with lipid and protein groups within cells, while the modes of drug action within the cells were assessed with the FLIM technique. RESULTS: Lensoside Aß itself does not exhibit anti-glioma activity but significantly enhances the anti-cancer potential of sorafenib, initiating mainly apoptosis of up to 90% of cells. It was correlated with an increased level of active caspase 3, a reduced MMP value, and a lower level of Raf kinase. The interaction with membrane structures led to morphological changes typical of programmed death. CONCLUSIONS: Our results indicate that lensoside Aß plays an important role as an adjuvant in chemotherapy with sorafenib and may be a potential candidate in anti-glioma combination therapy.

Involvement of PI3K Pathway in Glioma Cell Resistance to Temozolomide Treatment.

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.

Antifungal Activity of Anionic Defense Peptides: Insight into the Action of Galleria mellonella Anionic Peptide 2.

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Intracellular distribution of cadmium during the growth of Abortiporus biennis on cadmium-amended media.

Heavy metals are difficult to remediate and traditional remedial processes are expensive, so bioremediation technology using bacteria, fungi, or plants is of interest. Many studies have demonstrated that basidiomycetes fungi are able to growth under heavy metals stress. In this study the distribution of cadmium (Cd) in Abortiporus biennis cells was studied. Cd accumulated especially within cytoplasm and its presence caused changes in the cytoplasm appearance, which became denser in comparison to the cytoplasm of control cells. Vacuolization of cytoplasm and periplasmic region in A. biennis cells was also observed. The growth rate of A. biennis was inhibited up to 75% during the growth on medium amended with 1 mmol/L cadmium oxide. The presence of Cd in growing media inhibited oxalic acid secretion by A. biennis, but oxalate concentration increased together with elevated Cd concentration in growing medium. The influence of initial pH of growing media on the accumulation of Cd by A. biennis was also observed. The highest accumulation of Cd in mycelium was detected during A. biennis growth on media with a pH of 6. Studies addressing metals uptake by fungi and metal distribution in fungal cells may allow these organisms to be applied in bioremediation processes more effectively or to be used as bioindicators of contaminated environmental pollutions.

Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells.

Characteristics of quercetin interactions with liposomal and vacuolar membranes.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is claimed to exert many beneficial health effects. With application of (1)H NMR (nuclear magnetic resonance) and FTIR (Fourier-transform infrared) techniques, quercetin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was analyzed. Patch-clamp technique was employed to study quercetin effects at single channel level of vacuolar membranes in the liverwort Conocephalum conicum. Light and electron microscopy were applied to study quercetin effects on human negroid cervix carcinoma cells (HeLa). Enzymatic measurements along with DPPH (1,1-diphenyl-2-picrylhydrazyl) bioassay were performed to investigate the influence of quercetin on antioxidant enzymes and reactive oxygen species (ROS) production. The inclusion of quercetin to the membrane exerted pronounced ordering effect on the motional freedom of lipids in the head group region as manifested by broadening of the (1)H NMR spectral line representing the choline groups. FTIR analysis revealed quercetin incorporation into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C-O-P-O-C segment. Both, FTIR and NMR techniques indicated also quercetin spectral effects in the region corresponding to alkyl chains. Patch-clamp experiments showed that quercetin stabilizes tonoplast and promotes a close state of SV channels. Microscopic observations of HeLa cells revealed characteristic changes in ultrastructure and morphology of the examined cells in comparison to control cells. Pretreatment of HeLa cells with quercetin alleviated H2O2-induced cell injury by improving redox balance as indicated by the increase in glutathione content and SOD (superoxide dismutase) levels as well as by the decrease in ROS level. \In conclusion, the incorporation, distribution and the changes of biophysical properties of the membranes are very important for the effectiveness of phenolic compounds as antioxidant and anticancer factors.

FTIR, (1)H NMR and EPR spectroscopy studies on the interaction of flavone apigenin with dipalmitoylphosphatidylcholine liposomes.

Apigenin (5,7,4'-trihydroxyflavone) is a cancer chemopreventive agent and a member of the family of plant flavonoids. Apigenin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated by means of FTIR spectroscopy, (1)H NMR and EPR techniques. Fluorescent microscopy and electron microscopy were applied to study the apigenin effects on colon myofibroblasts and human skin fibroblasts. The strong rigidifying effect of apigenin with respect to polar head groups was concluded on the basis of the action of the flavone on partition coefficient of Tempo spin label between the water and lipid phases. The ordering effect was also found in hydrophobic region at the depth monitored by 5-SASL and 16-SASL spin labels. The inclusion of apigenin to the membrane restricted the motional freedom of polar head groups lowering penetration of Pr(3+) ions to the membranes. The (1)H NMR technique supported also the restriction of motional freedom of the membrane in the hydrophobic region, especially in the zone of CH(2) groups of alkyl chains. FTIR analysis showed that apigenin incorporates into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C-O-P-O-C segment. It is also very likely that hydroxyl groups of apigenin link with polar groups of DPPC by water bridges. Electron and fluorescence microscopic observations revealed changes in the internal membrane organization of the examined cells. In conclusion, the changes of the structural and dynamic properties of membranes can be crucial for processes involving tumor suppression signal transduction pathways and cell cycle regulation.

Anti-Legionella dumoffii activity of Galleria mellonella defensin and apolipophorin III.

The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires' disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 µg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins.

Localization and interaction of genistein with model membranes formed with dipalmitoylphosphatidylcholine (DPPC).

The effect of genistein on the liposomes formed with dipalmitoylphosphatidylcholine was studied with the application of Fourier-transform infrared spectroscopy, nuclear magnetic resonance (1H NMR) and electron paramagnetic resonance techniques. Membranous structures organization of human skin fibroblasts and colon myofibroblasts was also examined using fluorescence and electron microscopy. The strongest rigidifying effect of genistein with respect to polar head groups was concluded on the basis of the effect of the flavonoid on the shape of NMR lines attributed to -N+(CH3)3 groups. The rigidifying effect of genistein with respect to the hydrophobic core of lipid membranes was also concluded from the genistein-dependent broadening of the NMR lines assigned to -CH2 groups and terminal -CH3 groups of alkyl chains. EPR data supported ordering effect of genistein of the hydrophobic core in the liquid-crystalline phase (Lalpha). The analysis of the FTIR spectra of the two-component liposomes showed that genistein incorporates into DPPC membranes via hydrogen bonding between the lipid polar head groups in the C-O-P-O-C segment and its hydroxyl groups. Both fluorescence microscopy and ultrastructural observation revealed changes in membranous structures organization as aftermath of genistein treatment. In conclusion, genistein localized within membranes changes the properties of membrane that can be followed by the changes inside cells being crucial for pharmacological activity of genistein used in cancer or other disease treatment.

Genistein and changes of resting and action potentials in Conocephalum conicum.

Genistein (4',5,7-trixydroxyflavone) is a member of the family of plant flavonoids that widely occurs in crop plants. It is involved in a wide spectrum of pharmacological activities, and is suggested to have anti-cancer dietary properties. Cell membranes are one of the targets of anti-cancer drugs. In the present study, we used the liverwort Conocephalum conicum as a model plant in an electrophysiological study. Intracellular microelectrode measurements were carried out to examine the effects of genistein alone and in combination with verapamil on resting and action potentials. The application of isoflavone genistein resulted in a statistically significant elevation in action potential amplitudes. An increase of 13-62% compared with the control was noted. An increase was also found in the membrane resting potentials in genistein-treated plants. Verapamil, the known calcium channel inhibitor, caused a gradual decline of AP amplitudes, whereas preincubation of Conocephalum thalli with genistein prevented inhibition of APs by verapamil. It is concluded that genistein strongly affects the membranes, and the effect of genistein in canceling the activity of verapamil is discussed.