Attenuation of Nicotine Effects on A549 Lung Cancer Cells by Synthetic α7 nAChR Antagonists APS7-2 and APS8-2.

Nicotine binds to nicotinic acetylcholine receptors (nAChRs) that are overexpressed in different cancer cells, promoting tumor growth and resistance to chemotherapy. In this study, we aimed to investigate the potential of APS7-2 and APS8-2, synthetic analogs of a marine sponge toxin, to inhibit nicotine-mediated effects on A549 human lung cancer cells. Our electrophysiological measurements confirmed that APS7-2 and APS8-2 act as α7 nAChR antagonists. APS8-2 showed no cytotoxicity in A549 cells, while APS7-2 showed concentration-dependent cytotoxicity in A549 cells. The different cytotoxic responses of APS7-2 and APS8-2 emphasize the importance of the chemical structure in determining their cytotoxicity on cancer cells. Nicotine-mediated effects include increased cell viability and proliferation, elevated intracellular calcium levels, and reduced cisplatin-induced cytotoxicity and reactive oxygen species production (ROS) in A549 cells. These effects of nicotine were effectively attenuated by APS8-2, whereas APS7-2 was less effective. Our results suggest that APS8-2 is a promising new therapeutic agent in the chemotherapy of lung cancer.

Interaction of Nanomaterials with Cells and Tissues.

Nanomaterials have gained enormous importance in biomedicine in recent years, both in basic and applied sciences [...].

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, ß-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom ß-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity-In Vitro Correlation Analysis on THP-1 Macrophages.

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1ß cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1ß secretion. The induction of IL-1ß secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Disintegrins from the Venom of Vipera ammodytes ammodytes Efficiently Inhibit Migration of Breast Cancer Cells.

Integrins are plasma membrane proteins, whose dysfunction frequently results in cancer pathology, and therefore they represent important targets of anti-tumour therapy. Snake venoms are a rich source of disintegrins (Dis), proteins that specifically bind integrins and thus interfere with their functions. In an attempt to discover new molecules for treatment of breast cancer, the major type of cancer in women, we isolated a dimeric Dis (Vaa-Dis) from the venom of the nosehorned viper. By cell viability testing we demonstrated that 50 nM and higher concentrations of Vaa-Dis were toxic to highly invasive human breast adenocarcinoma cell line MDA-MB-231. Wound-healing assay revealed that already at one order of magnitude lower concentrations Vaa-Dis efficiently inhibited MDA-MB-231 cell migration. This exposed a promising anti-metastatic potential of Vaa-Dis and a good perspective of these natural snake venom proteins for further research and development towards the application in breast cancer treatment.

Time-dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes.

Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa488 ) and fluorescent dextran (Dextran546 ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa488 from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa488 . Initially, S100B-Alexa488 and Dextran546 internalized with distinct rates into different endocytotic vesicles; S100B-Alexa488 internalized into smaller vesicles than Dextran546 . At a later stage, S100B-Alexa488 -positive vesicles substantially co-localized with Dextran546 -positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa488 , but not of Dextran546 . The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa488 -positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca2+ ]i ). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs in vivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products.

Benzoic acid derivatives with improved antifungal activity: Design, synthesis, structure-activity relationship (SAR) and CYP53 docking studies.

Previously, we identified CYP53 as a fungal-specific target of natural phenolic antifungal compounds and discovered several inhibitors with antifungal properties. In this study, we performed similarity-based virtual screening and synthesis to obtain benzoic acid-derived compounds and assessed their antifungal activity against Cochliobolus lunatus, Aspergillus niger and Pleurotus ostreatus. In addition, we generated structural models of CYP53 enzyme and used them in docking trials with 40 selected compounds. Finally, we explored CYP53-ligand interactions and identified structural elements conferring increased antifungal activity to facilitate the development of potential new antifungal agents that specifically target CYP53 enzymes of animal and plant pathogenic fungi.

Structural insight into LexA-RecA* interaction.

RecA protein is a hallmark for the bacterial response to insults inflicted on DNA. It catalyzes the strand exchange step of homologous recombination and stimulates self-inactivation of the LexA transcriptional repressor. Importantly, by these activities, RecA contributes to the antibiotic resistance of bacteria. An original way to decrease the acquisition of antibiotic resistance would be to block RecA association with LexA. To engineer inhibitors of LexA-RecA complex formation, we have mapped the interaction area between LexA and active RecA-ssDNA filament (RecA*) and generated a three-dimensional model of the complex. The model revealed that one subunit of the LexA dimer wedges into a deep helical groove of RecA*, forming multiple interaction sites along seven consecutive RecA protomers. Based on the model, we predicted that LexA in its DNA-binding conformation also forms a complex with RecA* and that the operator DNA sterically precludes interaction with RecA*, which guides the induction of SOS gene expression. Moreover, the model shows that besides the catalytic C-terminal domain of LexA, its N-terminal DNA-binding domain also interacts with RecA*. Because all the model-based predictions have been confirmed experimentally, the presented model offers a validated insight into the critical step of the bacterial DNA damage response.

Ostreopexin: a hemopexin fold protein from the oyster mushroom, Pleurotus ostreatus.

Proteins with hemopexin repeats are widespread in viruses, prokaryotes and eukaryotes. We report here for the first time the existence of a protein in fungi with the four-bladed ß-propeller fold that is typical for hemopexin-like proteins. This protein was isolated from the edible basidiomycetous fungus Pleurotus ostreatus and is named ostreopexin. It binds to Ni(2+)-NTA-agarose, and is structurally and functionally very similar to PA2 albumins isolated from legume seeds and the hemopexin fold protein from rice. Like these plant proteins, ostreopexin shows reversible binding to hemin with moderate affinity, but does not bind to polyamines. We suggest that ostreopexin participates in intracellular management of metal (II or III)-chelates.

Chip electrophoretic separation of highly homologous ammodytoxin isoforms: three neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom.

Ammodytoxins (Atxs), a group of Ca(2+) -dependent neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom, are mainly responsible for venom toxicity. Within the Atx group, LD50 values between three isoforms, A, B, and C are differing with AtxA exhibiting an LD50 value by an order of magnitude lower (more toxic) than the other two isoforms. This difference in toxicity justifies the necessity to prepare suitable antibodies and thus isoform separation to characterize the Atx content of Vipera ammodytes ammodytes venom is of importance. However, a high homology between the three Atx isoforms (differences in only two, respectively, three residues within the last 18 amino acids at the C-terminus, total length 122 residues) hindered the successful separation of isoforms to date. As the investigated phospholipases A2 were reported to exhibit differences in pI values, we concentrate with the current work on the separation of Atx isoforms after fluorescence labeling via chip electrophoresis on a commercially available instrument to build the basis for a fast and easy to handle screening method. In the course of our work, we were able to show that samples of AtxA, AtxB, and AtxC declared to be homogenous by standard analytical techniques consisted indeed of more than one isoform of which the relative amounts were calculated by using the newly developed method.

Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

Targeted lipid analysis of haemolytic mycelial extracts of Aspergillus niger.

Ethanolic extracts of mycelia from Aspergillus niger (strain N402) grown in liquid media were observed to have haemolytic activity on bovine erythrocytes. This haemolytic activity decreased significantly during the time of growth (1-3 days). Moreover, when A. niger was grown on carbon-deprived medium, the efficiency of this haemolytic activity in the ethanolic extracts was much lower than when grown in carbon-enriched medium, and became almost undetectable after 3 days of growth in carbon-deprived medium. The lipid composition of these ethanolic extracts was analysed by liquid chromatography-electrospray ionisation tandem mass spectrometry. This haemolytic activity can be mainly linked to the relative levels of the molar ratios of the unsaturated fatty acids and lysophosphatidylcholines.

Gelatin nanoparticles loaded with 3-alkylpyridinium salt APS7, an analog of marine toxin, are a promising support in human lung cancer therapy.

This study demonstrates the potential of gelatin nanoparticles as a nanodelivery system for antagonists of nicotinic acetylcholine receptors (nAChRs) to improve chemotherapy efficacy and reduce off-target effects. Too often, chemotherapy for lung cancer does not lead to satisfactory results. Therefore, new approaches directed at multiple pharmacological targets in cancer therapy are being developed. Following the activation of nAChRs (e.g. by nicotine), cancer cells begin to proliferate and become more resistant to chemotherapy-induced apoptosis. This work shows that the 3-alkylpyridinium salt, APS7, a synthetic analog of a toxin from the marine sponge Haliclona (Rhizoneira) sarai, acts as an nAChR antagonist that inhibits the pro-proliferative and anti-apoptotic effects of nicotine on A549 human lung adenocarcinoma cells. In this study, gelatin-based nanoparticles filled with APS7 (APS7-GNPs) were prepared and their effects on A549 cells were compared with that of free APS7. Both APS7 and APS7-GNPs inhibited Ca2+ influx and increased the efficacy of cisplatin chemotherapy in nicotine-stimulated A549 cells. However, significant benefits from APS7-GNPs were observed - a stronger reduction in the proliferation of A549 lung cancer cells and a much higher selectivity in cytotoxicity towards cancer cells compared with non-tumorigenic lung epithelial BEAS-2B cells.

Improvement of fruit juice quality: novel endo-polygalacturonase II from Aspergillus tubingensis FAT 43 for enhanced liquefaction, clarification, and antioxidant potential.

This study focuses on the isolation, purification, and characterisation of endo-polygalacturonase II from Aspergillus tubingensis FAT43, particularly emphasising its potential applications in the fruit juice industry. A comprehensive screening test revealed the temporal dynamics of endo-polygalacturonase production during a 96-hour fermentation process. The purification process, involving ammonium sulfate and ethanol precipitation followed by ion-exchange chromatography, resulted in a 3.3-fold purification of PG II with a yield of 16% and a specific activity of 6001.67 U mg-1. Molecular analysis confirmed the identity of PG II, its gene (pgaII), and a high degree of sequence identity with Aspergillus tubingensis in the SWISS-PROT database. The optimal pH for PG II activity was 3.5-4.5, with robust stability across a broad pH spectrum (3-7). The enzyme exhibited optimal temperature activity at 45 °C, with a retention of 90% activity at 50 °C. The calculated activation energy for PG II was 62.1 kJ mol-1, indicating good stability. Inactivation kinetics revealed a half-life of 13.7 h at 40 °C, 5.4 h at 50 °C, and 0.85 h at 60 °C, with an activation energy of denaturation of 32.8 kJ mol-1. Compared to literature-reported PGs, PG II from A. tubingensis FAT43 demonstrated superior thermal stability. Hydrolysis experiments on different pectins revealed the highest specificity for non-methylated substrates (polygalacturonic acid). In fruit juice processing, PG II significantly increased juice yield and clarity, with the highest impact observed in strawberry juice. Antioxidant activity assays indicated enhanced antioxidant potential in enzyme-treated juices, especially strawberry, quince, and apple juices. The study highlights PG II's potential as an industrially valuable enzyme for fruit juice processing, offering improved thermostability and versatility across various fruit types.

The efficient magneto-mechanical actuation of cancer cells using a very low concentration of non-interacting ferrimagnetic hexaferrite nanoplatelets.

Magneto-mechanical actuation (MMA) using the low-frequency alternating magnetic fields (AMFs) of magnetic nanoparticles internalized into cancer cells can be used to irreparably damage these cells. However, nanoparticles in cells usually agglomerate, thus greatly augmenting the delivered force compared to single nanoparticles. Here, we demonstrate that MMA also decreases the cell viability, with the MMA mediated by individual, non-interacting nanoparticles. The effect was demonstrated with ferrimagnetic (i.e., permanently magnetic) barium-hexaferrite nanoplatelets (NPLs, ∼50 nm wide and 3 nm thick) with a unique, perpendicular orientation of the magnetization. Two cancer-cell lines (MDA-MB-231 and HeLa) are exposed to the NPLs in-vitro under different cell-culture conditions and actuated with a uniaxial AMF. TEM analyses show that only a small number of NPLs internalize in the cells, always situated in membrane-enclosed compartments of the endosomal-lysosomal system. Most compartments contain 1-2 NPLs and only seldom are the NPLs found in small groups, but never in close contact or mutually oriented. Even at low concentrations, the single NPLs reduce the cell viability when actuated with AMFs, which is further increased when the cells are in starvation conditions. These results pave the way for more efficient in-vivo MMA at very low particle concentrations.

Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds.

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.

Toxinology and Pharmacology of Snake Venoms.

Evolution endowed snakes with the ultimate weapon: venom [...].

Serine pseudoproteases in physiology and disease.

Serine proteases (SPs) constitute a very important family of enzymes, both physiologically and pathologically. The effects produced by these proteins have been explained by their proteolytic activity. However, the discovery of pharmacologically active SP molecules that show no enzymatic activity, as the so-called pseudo SPs or SP homologs (SPHs), has exposed a profoundly neglected possibility of nonenzymatic functions of these SP molecules. In this review, the most thoroughly described SPHs are presented. The main physiological domains in which SPHs operate appear to be in reproduction, embryonic development, immune response, host defense, and hemostasis. Hitherto unexplained actions of SPs should therefore be considered also as the result of the ligand-like attributes of SPs. The gain of a novel function by an SPH is a consequence of specific amino acid replacements that have resulted in a novel interaction interface or a 'catalytic trap'. Unraveling the SP/SPH interactome will provide a description of previously unknown physiological functions of SPs/SPHs, aiding the creation of innovative medical approaches.