Apoptosis and eryptosis: similarities and differences.

Eryptosis is a regulated cell death (RCD) of mature erythrocytes initially described as a counterpart of apoptosis for enucleated cells. However, over the recent years, a growing number of studies have emphasized certain differences between both cell death modalities. In this review paper, we underline the hallmarks of eryptosis and apoptosis and highlight resemblances and dissimilarities between both RCDs. We summarize and critically discuss differences in the impact of caspase-3, Ca2+ signaling, ROS signaling pathways, opposing roles of casein kinase 1α, protein kinase C, Janus kinase 3, cyclin-dependent kinase 4, and AMP-activated protein kinase to highlight a certain degree of divergence between apoptosis and eryptosis. This review emphasizes the crucial importance of further studies that focus on deepening our knowledge of cell death machinery and identifying novel differences between cell death of nucleated and enucleated cells. This might provide evidence that erythrocytes can be defined as viable entities capable of programmed cell destruction. Additionally, the revealed cell type-specific patterns in cell death can facilitate the development of cell death-modulating therapeutic agents.

Erythronecroptosis: an overview of necroptosis or programmed necrosis in red blood cells.

Necroptosis is considered a programmed necrosis that requires receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and pore-forming mixed lineage kinase domain-like protein (MLKL) to trigger a regulated cell membrane lysis. Membrane rupture in necroptosis has been shown to fuel innate immune response due to release of damage-associated molecular patterns (DAMPs). Recently published studies indicate that mature erythrocytes can undergo necroptosis as well. In this review, we provide an outline of multiple cell death modes occurring in erythrocytes, discuss possible immunological aspects of diverse erythrocyte cell deaths, summarize available evidence related to the ability of erythrocytes to undergo necroptosis, outline key involved molecular mechanisms, and discuss the potential implication of erythrocyte necroptosis in the physiology and pathophysiology. Furthermore, we aim to highlight the interplay between necroptosis and eryptosis signaling in erythrocytes, emphasizing specific characteristics of these pathways distinct from their counterparts in nucleated cells. Thus, our review provides a comprehensive summary of the current knowledge of necroptosis in erythrocytes. To reflect critical differences between necroptosis of nucleated cells and necroptosis of erythrocytes, we suggest a term erythronecroptosis for necroptosis of enucleated cells.

Antibacterial and antioxidant activity of gold and silver nanoparticles in dextran-polyacrylamide copolymers.

Search for new antimicrobial agents is of great significance due to the issue of antimicrobial resistance, which nowadays has become more important than many diseases. The aim of this study was to evaluate the toxicity and biological effects of a dextran-graft-polyacrylamide (D-PAA) polymer-nanocarrier with/without silver or gold nanoparticles (AgNPs/D-PAA and AuNPs/D-PAA, respectively) to analyze their potential to replace or supplement conventional antibiotic therapy. The toxicity of nanocomplexes against eukaryotic cells was assessed on primary dermal fibroblasts using scratch, micronucleus and proliferation assays. DPPH (2,2-diphenyl-1-picrylhydrazylradical) assay was used to evaluate the antioxidant capacity of D-PAA, AgNPs/D-PAA and AuNPs/D-PAA. DNA cleavage, antimicrobial and biofilm inhibition effects of nanocomplexes were investigated. Nanocomplexes were found to be of moderate toxicity against fibroblasts with no genotoxicity observed. AgNPs/D-PAA reduced motility and proliferation at lower concentrations compared with the other studied nanomaterials. AgNPs/D-PAA and AuNPs/D-PAA showed radical scavenging capacities in a dose-dependent manner. The antimicrobial activity of AgNPs/D-PAA against various bacteria was found to be much higher compared to D-PAA and AuNPs/D-PAA, especially against E. hirae, E. faecalis and S. aureus, respectively. D-PAA, AgNPs/D-PAA and AuNPs/D-PAA showed DNA-cleaving and biofilm inhibitory activity, while AgNPs/D-PAA displayed the highest anti-biofilm activity. AgNPs/D-PAA and AuNPs/D-PAA were characterized by good antimicrobial activity. According to the findings of the study, AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of new antimicrobial agents, the fight against biofilms, sterilization and disinfection processes. Our findings confirm the versatility of nanosystems based on dextran-polyacrylamide polymers and indicate that AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of novel antimicrobial agents.

Casein kinase 1α mediates eryptosis: a review.

Eryptosis is a coordinated non-lytic cell death of erythrocytes characterized by cell shrinkage, cell membrane scrambling, Ca2+ influx, ceramide accumulation, oxidative stress, activation of calpain and caspases. Physiologically, it aims at removing damaged or aged erythrocytes from circulation. A plethora of diseases are associated with enhanced eryptosis, including metabolic diseases, cardiovascular pathology, renal and hepatic diseases, hematological disorders, systemic autoimmune pathology, and cancer. This makes eryptosis and eryptosis-regulating signaling pathways a target for therapeutic interventions. This review highlights the eryptotic signaling machinery containing several protein kinases and its small molecular inhibitors with a special emphasis on casein kinase 1α (CK1α), a serine/threonine protein kinase with a broad spectrum of activity. In this review article, we provide a critical analysis of the regulatory role of CK1α in eryptosis, highlight triggers of CK1α-mediated suicidal death of red blood cells, cover the knowledge gaps in understanding CK1α-driven eryptosis and discover the opportunity of CK1α-targeted pharmacological modulation of eryptosis. Moreover, we discuss the directions of future research focusing on uncovering crosstalks between CK1α and other eryptosis-regulating kinases and pathways.

Hemocompatibility of dextran-graft-polyacrylamide/zinc oxide nanosystems: hemolysis or eryptosis?

Aim. In this study, blood compatibility of ZnO nanoparticles-polymer nanocomplex (D-PAA/ZnONPs(SO42-)) synthesizedin situinto dextran-graft-polyacrylamide (D-PAA) using zinc sulphate as a precursor was tested using hemolysis, osmotic fragility and eryptosis assays.Materials and methods. Dose-dependent ability to induce eryptosis was assessed following 24 h incubation at concentrations of 0-800 mg l-1analyzing hallmarks of eryptosis (cell shrinkage and phosphatidylserine externalization), as well as reactive oxygen species generation. Hemolysis was detected spectrophotometrically based on hemoglobin release following exposure to the D-PAA/ZnONPs(SO42-) nanocomplex. Osmotic fragility test (OFT) involved detection of hemolysis of red blood cells exposed to 0.2% saline solution following incubation with the D-PAA/ZnONPs(SO42-) nanocomplex. Additional incubation of the nanocomplex in the presence or absence of either ascorbic acid or EGTA was used to reveal the implication of oxidative stress- or Ca2+-mediated mechanisms in D-PAA/ZnONPs(SO42-) nanocomplex-induced erythrotoxicity.Results. Hemocompatibility assessment of the D-PAA/ZnONPs(SO42-) nanocomplex revealed that it induced hemolysis and reduced resistance of erythrocytes to osmotic stress at concentrations of above 400 and 200 mg l-1, respectively. Oxidative stress- or Ca2+-mediated mechanisms were not involved in D-PAA/ZnONPs(SO42-) nanocomplex-induced hemolysis. Strikingly, the D-PAA/ZnONPs(SO42-) nanocomplex did not promote cell membrane scrambling, cell shrinkage and oxidative stress in red blood cells following the direct exposure for 24 h. Thus, the D-PAA/ZnONPs(SO42-) nanocomplex did not induce eryptosisin vitro. Eryptosis is generally considered to occur earlier than hemolysis in response to stress in order to prevent hemolytic cell death. Counterintuitively, our data suggest that hemolysis can be triggered by nanomaterials prior to eryptosis indicating that eryptosis and hemolysis assays should be used in combination for testing blood compatibility of nanomaterials.Conclusions. The D-PAA/ZnONPs(SO42-) nanocomplex has a good hemocompatibility profile at low concentrations. Hemocompatibility testing in nanotoxicology should include both eryptosis and hemolysis assays.

Rare-earth orthovanadate nanoparticles trigger Ca2+-dependent eryptosis.

Introduction. Rare-earth orthovanadate nanoparticles (ReVO4:Eu3+, Re = Gd, Y or La) are promising agents for photodynamic therapy of cancer due to their modifiable redox properties. However, their toxicity limits their application.Objective. The aim of this research was to elucidate pro-eryptotic effects of GdVO4:Eu3+and LaVO4:Eu3+nanoparticles with identification of underlying mechanisms of eryptosis induction and to determine their pharmacological potential in eryptosis-related diseases.Methods. Blood samples (n= 9) were incubated for 24 h with 0-10-20-40-80 mg l-1GdVO4:Eu3+or LaVO4:Eu3+nanoparticles, washed and used to prepare erythrocyte suspensions to analyze the cell membrane scrambling (annexin-V-FITC staining), cell shrinkage (forward scatter signaling), reactive oxygen species (ROS) generation through 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining and intracellular Ca2+levels via FLUO4 AM staining by flow cytometry. Internalization of europium-enabled luminescent GdVO4:Eu3+and LaVO4:Eu3+nanoparticles was assessed by confocal laser scanning microscopy.Results.Both nanoparticles triggered eryptosis at concentrations of 80 mg l-1. ROS-mediated mechanisms were not involved in rare-earth orthovanadate nanoparticles-induced eryptosis. Elevated cytosolic Ca2+concentrations were revealed even at subtoxic concentrations of nanoparticles. LaVO4:Eu3+nanoparticles increased intracellular calcium levels in a more pronounced way compared with GdVO4:Eu3+nanoparticles. Our data disclose that the small-sized (15 nm) GdVO4:Eu3+nanoparticles were internalized after a 24 h incubation, while the large-sized (∼30 nm) LaVO4:Eu3+nanoparticles were localized preferentially around erythrocytes.Conclusions.Both internalized GdVO4:Eu3+and non-internalized LaVO4:Eu3+nanoparticles (80 mg l-1) promote eryptosis of erythrocytes after a 24 h exposurein vitrovia Ca2+signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.

Redox Status of Erythrocytes as an Important Factor in Eryptosis and Erythronecroptosis.

Overall, reactive oxygen species (ROS) signalling significantly contributes to initiation and mo-dulation of multiple regulated cell death (RCD) pathways. Lately, more information has become available about RCD modalities of erythrocytes, including the role of ROS. ROS accumulation has therefore been increasingly recognized as a critical factor involved in eryptosis (apoptosis of erythrocytes) and erythro-necroptosis (necroptosis of erythrocytes). Eryptosis is a Ca2+-dependent apoptosis-like RCD of erythrocytes that occurs in response to oxidative stress, hyperosmolarity, ATP depletion, and a wide range of xenobiotics. Moreover, eryptosis seems to be involved in the pathogenesis of multiple human diseases and pathological processes. Several studies have reported that erythrocytes can also undergo necroptosis, a lytic RIPK1/RIPK3/MLKL-mediated RCD. As an example, erythronecroptosis can occur in response to CD59-specific pore-forming toxins. We have systematically summarized available studies regarding the involvement of ROS and oxidative stress in these two distinct RCDs of erythrocytes. We have focused specifically on cellular signalling pathways involved in ROS-mediated cell death decisions in erythrocytes. Furthermore, we have summarized dysregulation of related erythrocytic antioxidant defence systems. The general concept of the ROS role in eryptotic and necroptotic cell death pathways in erythrocytes seems to be established. However, further studies are required to uncover the complex role of ROS in the crosstalk and interplay between the survival and RCDs of erythrocytes.

B-Cell Receptor Signaling and Beyond: The Role of Igα (CD79a)/Igß (CD79b) in Normal and Malignant B Cells.

B-cell receptor (BCR) is a B cell hallmark surface complex regulating multiple cellular processes in normal as well as malignant B cells. Igα (CD79a)/Igß (CD79b) are essential components of BCR that are indispensable for its functionality, signal initiation, and signal transduction. CD79a/CD79b-mediated BCR signaling is required for the survival of normal as well as malignant B cells via a wide signaling network. Recent studies identified the great complexity of this signaling network and revealed the emerging role of CD79a/CD79b in signal integration. In this review, we have focused on functional features of CD79a/CD79b, summarized signaling consequences of CD79a/CD79b post-translational modifications, and highlighted specifics of CD79a/CD79b interactions within BCR and related signaling cascades. We have reviewed the complex role of CD79a/CD79b in multiple aspects of normal B cell biology and how is the normal BCR signaling affected by lymphoid neoplasms associated CD79A/CD79B mutations. We have also summarized important unresolved questions and highlighted issues that remain to be explored for better understanding of CD79a/CD79b-mediated signal transduction and the eventual identification of additional therapeutically targetable BCR signaling vulnerabilities.

THE LEVEL OF REACTIVE OXYGEN SPECIES AS A MARKER OF ASTHMA SEVERITY IN CHILDREN.

OBJECTIVE: The aim of the research was to assess the reactive oxygen species (ROS) levels in granulocytes of patients with asthma. PATIENTS AND METHODS: Materials and methods: The study involved 35 children aged 5 to 17 years. 26 children with persistent asthma, partially controlled course in the period of exacerbation were divided into groups: 1 group - mild asthma (n = 12), group 2 - moderate asthma (n = 7) group 3 - severe asthma (n = 7) and control group included almost healthy children (n = 9). ROS levels in granulocytes were evaluated using BD FACSDiva™. The spirographic complex was used to assess the function of external respiration. RESULTS: Results: The level of ROS in granulocytes of patients with severe asthma was significantly reduced compared with children in the control group and patients with mild and moderate asthma (p1-3 = 0.0003, p2-3 = 0.0017, p c-3 = 0.0150). The concentration of ROS in granulocytes ≤ 285 a.u. was prognostically significant with high specificity and sensitivity with severe asthma. CONCLUSION: Conclusions: The concentration of ROS levels in neutrophils in patients with severe asthma probably reflected the suppression of their products, which suggests the depletion of the reserve capacity of neutrophils. Decreased concentrations of reactive oxygen species in children with asthma can be considered as a possible marker of asthma severity.

Antimicrobial Effects of Nanostructured Rare-Earth-Based Orthovanadates.

The search for novel antimicrobial agents is of huge importance. Nanomaterials can come to the rescue in this case. The aim of this study was to assess the cytotoxicity and antimicrobial effects of rare-earth-based orthovanadate nanoparticles. The cytotoxicity against host cells and antimicrobial activity of LaVO4:Eu3+ and GdVO4:Eu3+ nanoparticles were analyzed. Effects of nanomaterials on fibroblasts were assessed by MTT, neutral red uptake and scratch assays. The antimicrobial effects were evaluated by the micro-dilution method estimating the minimum inhibitory concentration (MIC) of nanoparticles against various strains of microorganisms, DNA cleavage and biofilm inhibition. GdVO4:Eu3+ nanoparticles were found to be less toxic against eukaryotic cells compared with LaVO4:Eu3+. Both nanoparticles exhibited antimicrobial activity and the highest MIC values were 64 mg/L for E. hirae, E. faecalis and S. aureus shown by GdVO4:Eu3+ nanoparticles. Nanoparticles demonstrated good DNA cleavage activity and induction of double-strand breaks in supercoiled plasmid DNA even at the lowest concentrations used. Both nanoparticles showed the biofilm inhibition activity against S. aureus at 500 mg/L and reduced the microbial cell viability. Taken the results of host toxicity and antimicrobial activity studies, it can be assumed that GdVO4:Eu3+ nanoparticles are more promising antibacterial agents compared with LaVO4:Eu3+ nanoparticles.

Effectiveness of inhaled hypertonic saline application for sputum induction to improve Mycobacterium tuberculosis identification in patients with pulmonary tuberculosis.

BACKGROUND: This study assessed the effectiveness and diagnostic significance of hypertonic saline sputum induction for improving Mycobacterium tuberculosis (MTB) detection. METHODS: A prospective, randomized, open, two-arm, comparative study on MTB identification effectiveness when using inhaled sodium chloride hypertonic solution was performed in patients diagnosed with pulmonary tuberculosis (TB). Patients were randomly assigned into two groups: group 1 (inhalation group) included patients who inhaled a 7% sodium chloride solution upon admission to the hospital, and group 2 (control group) coughed up their sputum as usual. For both groups, specimens were tested by bacterioscopic, bacteriological, and molecular genetic methods. Diagnostic chest radiography was performed for all participants. RESULTS: In this study, 644 patients (mean age 42.2 years; 151 women, 23.4%) were randomly divided into two groups. Low-quality sputum samples were observed in 7.4% of patients from the inhalation group and 28.8% in the control group (p < 0.001). Acid-fast bacilli (AFB) smear was positive in 65.1% of patients from the inhalation group and 51.3% of controls (p = 0.002). A similar statistically significant situation was observed when culture methods (93.9% inhalation group and 81.9% control group, p < 0.001) and molecular genetic tests (92.2% inhalation group and 79.4% control group, p < 0.001) were used. Thus, active pulmonary TB was not verified microbiologically in 6.1% of patients from the inhalation group and in 18.1% of controls (p < 0.001). CONCLUSIONS: Hypertonic saline sputum induction improves the quality of collected samples. This method may be appropriate to increase the rate of MTB detection in sputum using microscopic, bacteriological, and molecular genetic methods for diagnosing TB on the day of specimen collection. Hypertonic saline sputum induction is suitable for middle- and low-income countries with limited resources and causes no severe adverse effects in TB patients.

Experimental confirmation of antimicrobial effects of GdYVO4:Eu3+ nanoparticles.

Nanotechnology can be applied to design antibacterial agents to combat antibiotic resistance. The aim of the present study was to assess the antimicrobial effects and cytotoxicity of GdYVO4:Eu3+ nanoparticles (NPs). Biofilm inhibition activity, antimicrobial activity, bacterial viability inhibition and DNA cleavage activity of GdYVO4:Eu3+ NPs were studied. In addition, the impact of GdYVO4:Eu3+ NPs on the mitochondrial membrane potential (ΔΨM) of host immune cells and, hence, their apoptosis was analyzed by JC-1 staining using flow cytometry. GdYVO4:Eu3+ NPs demonstrated good antimicrobial, cell viability inhibition and DNA cleavage activities. In addition, GdYVO4:Eu3+ NPs showed good biofilm inhibition activity against S. aureus and P. aeruginosa and inhibition percentages were 89.15% and 79.54%, respectively. However, GdYVO4:Eu3+ NPs promoted mitochondrial depolarization and apoptosis of leukocytes at high concentrations. GdYVO4:Eu3+ nanoparticles are promising antibacterial agents. However, more efforts should be exerted to ensure their safety.

Experimental Evaluation of Food-Grade Semi-Refined Carrageenan Toxicity.

The safety of food additives E407 and E407a has raised concerns in the scientific community. Thus, this study aims to assess the local and systemic toxic effects of the common food additive E407a in rats orally exposed to it for two weeks. Complex evaluations of the effects of semi-refined carrageenan (E407a) on rats upon oral exposure were performed. Local effects of E407a on the intestine were analyzed using routine histological stains and CD68 immunostaining. Furthermore, circulating levels of inflammatory markers were assessed. A fluorescent probe O1O (2- (2'-OH-phenyl)-5-phenyl-1,3-oxazole) was used for evaluating the state of leukocyte cell membranes. Cell death modes of leukocytes were analyzed by flow cytometry using Annexin V and 7-aminoactinomycin D staining. Oral administration of the common food additive E407a was found to be associated with altered small and large intestinal morphology, infiltration of the lamina propria in the small intestine with macrophages (CD68+ cells), high systemic levels of inflammation markers, and changes in the lipid order of the phospholipid bilayer in the cell membranes of leukocytes, alongside the activation of their apoptosis. Our findings suggest that oral exposure to E407a through rats results in the development of intestinal inflammation.

Food additive E407a stimulates eryptosis in a dose-dependent manner.

BACKGROUND: Concerns about the biosafety of the common food additive E407a have been raised. It has been demonstrated to induce intestinal inflammation, accompanied by activation of apoptosis, upon oral exposure. Thus, it is of interest to investigate how E407a affects eryptosis, a suicidal cell death mode of red blood cells. OBJECTIVE: To evaluate the effects of semi-refined carrageenan (E407a) on eryptosis. METHODS: Flow cytometry was employed to assess eryptosis in blood exposed to various concentrations of E407a (0 g/L, 1 g/L, 5 g/L, and 10 g/L) during incubation for 24 h by analyzing phosphatidylserine externalization in erythrocytes using annexin V staining and via evaluating reactive oxygen species (ROS) generation using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In addition, the eryptosis indices mentioned above were determined in rats orally administered E407a at a dose of 140 mg/kg weight for 2 weeks. Confocal scanning laser microscopy was performed to visualize cell membrane scrambling. RESULTS: Oral intake of E407a for 2 weeks by rats was not associated with membrane scrambling in erythrocytes. However, ROS overproduction was observed. Meanwhile, incubation of blood with various concentrations of semi-refined carrageenan resulted in a dose-dependent promotion of eryptosis, evidenced by the enhanced percentage of annexin V-positive erythrocytes and higher mean fluorescence intensity (MFI) values of annexin V-FITC in all erythrocytes. The highest concentration of E407a promotes a statistically significant increase in ROS generation in erythrocytes, suggesting the role of ROS-mediated induction of eryptosis in this case. CONCLUSION: Incubation of blood with the food additive E407a leads to the activation of eryptosis in a dose-dependent manner. ROS-mediated mechanisms are partially responsible for E407a-induced eryptosis.

Semi-refined carrageenan promotes generation of reactive oxygen species in leukocytes of rats upon oral exposure but not in vitro.

AIM: To assess the ability of the common food additive E407a (semi-refined carrageenan) to enter leukocytes in vitro and generate reactive oxygen species (ROS) in leukocytes as a whole and granulocytes in particular, both during incubation and in experimental animals. METHODS: ROS production was assessed in leukocytes incubated with E407a for 2 h at the final concentrations of 5 and 10 g/L using the dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as in cells isolated from rats orally exposed to E407a (140 mg/kg of weight) during 2 weeks (n = 8) and control rats (n = 8), by flow cytometry. Carrageenan uptake by leukocytes was estimated by confocal microscopy using incubation of rhodamine B isothiocyanate-labelled carrageenan with leukocyte suspensions. RESULTS: Uptake of carrageenan by viable neutrophils, monocytes, and lymphocytes was confirmed. Oral administration of the food additive E407a was associated with excessive ROS formation by viable leukocytes (CD45+, 7­aminoactinomycin D- cells) and especially in granulocytes. Unexpectedly, a direct impact of semi-refined carrageenan during incubation for 2 h did not affect ROS production in leukocytes, evidenced by statistically insignificant differences in mean fluorescence intensity values of 2',7'-dichlorofluorescein, which is a ROS-sensitive product of intracellular H2DCFDA conversion. Oral intake of E407a and direct exposure of leukocyte suspensions to it decreased the viability of leukocytes. CONCLUSION: Food-grade carrageenan can enter leukocytes without affecting ROS generation as a result of incubation for 2 h with leukocyte suspensions. On the contrary, oral exposure to E407a is accompanied by ROS overproduction by white blood cells, suggesting an indirect mechanism for the stimulation of ROS synthesis in vivo. E407a promotes cell death of leukocytes both in vivo and in vitro.

Orally administered gadolinium orthovanadate GdVO4:Eu3+ nanoparticles do not affect the hydrophobic region of cell membranes of leukocytes.

AIM: To assess the phospholipid bilayer of white blood cells (WBCs) and the ability of leukocytes to generate reactive oxygen species (ROS) in rats orally exposed to GdVO4:Eu3+ nanoparticle (VNP) solution for 2 weeks by fluorescent probes-ortho-hydroxy derivatives of 2,5-diaryl­1,3­oxazole. METHODS: Steady-state fluorescence spectroscopy, i.e., a study by the environment-sensitive fluorescent probes 2­(2'-OH-phenyl)-5-(4'-phenyl-phenyl)-1,3-oxazole (probe O6O) and 2­(2'-OH-phenyl)-phenanthro[9,10]-1,3-oxazole (probe PH7), and flow cytometry, i.e., analysis of 2',7'-dichlorofluorescein (DCF), a product of a dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), fluorescence in CD45+/7-aminoactinomycin D (7-AAD)- cells, were used to evaluate the state of cell membranes and reactive oxygen species (ROS) generation in leukocytes of rats orally exposed to gadolinium orthovanadate nanoparticles(VNPs). RESULTS: No significant changes were detected in the spectra of the fluorescent probes bound to the WBCs from the rats orally exposed to nanoparticles in comparison with the corresponding spectra of the probes bound to the cells from the control group of animals. This indicates that in the case of the rats orally exposed to nanoparticles, no noticeable changes in physicochemical properties (i.e., in the polarity and the proton-donor ability) are observed in the lipid membranes of WBCs in the region where the probes locate. There was no statistically significant difference in the amount of ROShigh viable leukocytes in rats treated with VNPs and control samples. CONCLUSION: Neither changes in the physical and chemical properties of the leukocyte membranes nor in ROS generation by WBCs are detected in the rats orally exposed to VNP solution for 2 weeks.

Diphytanoyl lipids as model systems for studying membrane-active peptides.

The branched chains in diphytanoyl lipids provide membranes with unique properties, such as high chemical/physical stability, low water permeability, and no gel-to-fluid phase transition at ambient temperature. Synthetic diphytanoyl phospholipids are often used as model membranes for electrophysiological experiments. To evaluate whether these sturdy lipids are also suitable for solid-state NMR, we have examined their interactions with a typical amphiphilic peptide in comparison with straight-chain lipids. First, their phase properties were monitored using 31P NMR, and the structural behaviour of the antimicrobial peptide PGLa was studied by 19F NMR and circular dichroism in oriented membrane samples. Only lipids with choline headgroups (DPhPC) were found to form stable lipid bilayers in oriented samples, while DPhPG, DPhPE and DPhPS display non-lamellar structures. Hence, the experimental temperature and hydration are crucial factors when using supported diphytanoyl lipids, as both parameters must be maintained in an appropriate range to avoid the formation of non-bilayer structures. For the same reason, a high content of other diphytanoyl lipids besides DPhPC in mixed lipid systems is not favourable. Unlike the situation in straight-chain membranes, we found that the α-helical PGLa was not able to insert into the tightly packed fluid bilayer of DPhPC but remained in a surface-bound state even at very high peptide concentration. This behaviour can be explained by the high cohesivity and the negative spontaneous curvature of the diphytanoyl lipids. These characteristic features must therefore be taken into consideration, both, in electrophysiological studies, and when interpreting the structural behaviour of membrane-active peptides in such lipid environment.