Search

Purine nucleotide synthesis is realised only through the salvage pathway in pathogenic bacterium Helicobacter pylori. Therefore, the enzymes of this pathway, among them also the adenylosuccinate synthetase (AdSS), present potential new drug targets. This paper describes characterization of His6-tagged AdSS from H. pylori. Thorough analysis of 3D-structures of fully ligated AdSS (in a complex with guanosine diphosphate, 6-phosphoryl-inosine monophosphate, hadacidin and Mg2+) and AdSS in a complex with inosine monophosphate (IMP) only, enabled identification of active site interactions crucial for ligand binding and enzyme activity. Combination of experimental and molecular dynamics (MD) simulations data, particularly emphasized the importance of hydrogen bond Arg135-IMP for enzyme dimerization and active site formation. The synergistic effect of substrates (IMP and guanosine triphosphate) binding was suggested by MD simulations. Several flexible elements of the structure (loops) are stabilized by the presence of IMP alone, however loops comprising residues 287–293 and 40–44 occupy different positions in two solved H. pylori AdSS structures. MD simulations discovered the hydrogen bond network that stabilizes the closed conformation of the residues 40–50 loop, only in the presence of IMP. Presented findings provide a solid basis for the design of new AdSS inhibitors as potential drugs against H. pylori.

Alicja Hinz,1 Marta Szczęch,2 Krzysztof Szczepanowicz,2 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Kraków, PolandCorrespondence: Monika BzowskaDepartment of Cell Biochemistry, Faculty of Biochemistry, Jagiellonian University in Kraków, Biophysics and Biotechnology, 7 Gronostajowa Street, Kraków, 30-387, Poland, Tel/Fax +48 12 664 63 88, Email monika.bzowska@uj.edu.plKrzysztof SzczepanowiczJerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8 Street, Kraków, 30-239, Poland, Tel/Fax +48 12 639 51 21, Email krzysztof.szczepanowicz@ikifp.edu.plIntroduction: Biodistribution of nanocarriers with a structure consisting of core and shell is most often analyzed using methods based on labeling subsequent compartments of nanocarriers. This approach may have serious limitations due to the instability of such complex systems under in vivo conditions.Methods: The core-shell polyelectrolyte nanocarriers were intravenously administered to healthy BALB/c mice with breast cancer. Next, biodistribution profiles and elimination routes were determined post mortem based on fluorescence measurements performed for isolated blood, tissue homogenates, collected urine, and feces.Results: Despite the surface PEGylation with PLL-g-PEG, multilayer polyelectrolyte nanocarriers undergo rapid degradation after intravenous administration. This process releases the shell components but not free Rhodamine B. Elements of polyelectrolyte shells are removed by hepatobiliary and renal clearance.Conclusion: Multilayer polyelectrolyte nanocarriers are prone to rapid degradation after intravenous administration. Fluorophore localization determines the obtained results of biodistribution and elimination routes of core-shell nanomaterials. Therefore, precise and reliable analysis of in vivo stability and biodistribution of nanomaterials composed of several compartments requires nanomaterials labeled within each compartment.Keywords: in vivo studies, fluorescently labeled nanomaterials, stealth polymers, pharmacokinetics, routes of elimination, nanocarriers stability and degradation

Objective and design BacSp222 bacteriocin is a bactericidal and proinflammatory peptide stimulating immune cells to produce selected cytokines and NO in NF-ĸB dependent manner. This study aims to identify the receptor which mediates this activity. Methods We applied fluorescently labeled BacSp222 and a confocal microscopy imaging to analyze the direct interaction of the bacteriocin with the cells. Reporter HEK-Blue cells overexpressing human toll-like receptors (TLR2, TLR4, TLR5 or TLR2/TLR1 and TLR2/TLR6 heterodimers) were stimulated with BacSp222, and then the activity of NF-ĸB-dependent secreted embryonic alkaline phosphatase (SEAP) was measured. In turn, formylated peptide receptor (FPR) or TLR2 antagonists were used to verify bacteriocin-stimulated TNF production by murine monocyte-macrophage cell lines. Results BacSp222 undergoes internalization into cells without disturbing the cell membrane. FPR antagonists do not affect TNF produced by BacSp222-stimulated murine macrophage-like cells. In contrast, BacSp222 stimulates NF-ĸB activation in HEK-Blue overexpressing TLR2 or TLR2/TLR6 heterodimer, but not TLR2/TLR1, TLR4 or TLR5 receptors. Moreover, TLR2-specific antagonists inhibit NF-ĸB signaling in BacSp222-stimulated HEK-Blue TLR2/TLR6 cells and reduce TNF release by BacSp222-treated RAW 264.7 and P388.D1. Conclusions BacSp222 is a novel ligand for TLR2/TLR6 heterodimer. By binding TLR complex the bacteriocin undergoes internalization, inducing proinflammatory signaling that employs MyD88 and NF-ĸB pathways.

Novel multifunctional biomimetic injectable hybrid systems were synthesized. The physicochemical as well as biological in vitro and in vivo tests demonstrated that they are promising candidates for bone tissue regeneration. The hybrids are composed of a biopolymeric collagen/chitosan/hyaluronic acid matrix and amine group-functionalized silica particles decorated with apatite to which the alendronate molecules were coordinated. The components of these systems were integrated and stabilized by cross-linking with genipin, a compound of natural origin. They can be precisely injected into the diseased tissue in the form of a viscous sol or a partially cross-linked hydrogel, where they can serve as scaffolds for locally controlled bone tissue regeneration/remodeling by supporting the osteoblast formation/proliferation and maintaining the optimal osteoclast level. These materials lack systemic toxicity. They can be particularly useful for the repair of small osteoporotic bone defects.

Dementia is one of the major health issues around the globe which can severely affect the daily functioning of patients and their caregivers. There is no pharmacological method that entirely cures dementia, however, early detection, precognitive medication and treatment of concomitant illnesses and behavioural symptoms can significantly moderate cognitive decline as well as improve the quality of life patients with dementia. Here we present a case of Dementia with Lewy Bodies (DLB), which is the second most common form of neurodegenerative dementia in population above 65 years old. Despite its common occurrence, this disease is rarely detected, due to misleading symptoms, in its early stage. DLB often starts with depressive or psychotic symptoms, whilst cognitive decline is not apparent yet. Clinical features of DLB, as well as challenges of diagnosis and management are presented accordingly.

The rational design of novel self-assembled nanomaterials based on peptides remains a great challenge in modern chemistry. A hierarchical approach for the construction of nanofibrils based on α,β-peptide foldamers is proposed. The incorporation of a helix-promoting trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue in the outer positions of the model coiled-coil peptide led to its increased conformational stability, which was established consistently by the results of CD, NMR and FT-IR spectroscopy. The designed oligomerization state in the solution of the studied peptides was confirmed using analytical ultracentrifugation. Moreover, the cyclopentane side chain allowed additional interactions between coiled-coil-like structures to direct the self-assembly process towards the formation of well-defined nanofibrils, as observed using AFM and TEM techniques.

Polymeric nanomaterials have become a prominent area of research in the field of drug delivery. Their application in nanomedicine can improve bioavailability, pharmacokinetics, and, therefore, the effectiveness of various therapeutics or contrast agents. There are many studies for developing new polymeric nanocarriers; however, their clinical application is somewhat limited. In this review, we present new complex and multifunctional polymeric nanocarriers as promising and innovative diagnostic or therapeutic systems. Their multifunctionality, resulting from the unique chemical and biological properties of the polymers used, ensures better delivery, and a controlled, sequential release of many different therapeutics to the diseased tissue. We present a brief introduction of the classical formulation techniques and describe examples of multifunctional nanocarriers, whose biological assessment has been carried out at least in vitro. Most of them, however, also underwent evaluation in vivo on animal models. Selected polymeric nanocarriers were grouped depending on their medical application: anti-cancer drug nanocarriers, nanomaterials delivering compounds for cancer immunotherapy or regenerative medicine, components of vaccines nanomaterials used for topical application, and lifestyle diseases, ie, diabetes.

The implementation of precision farming technologies into agricultural practice requires, among other things, precise determination of the extent and intensity of insect infestation in the farmer' fields. Manual insect identification is time-consuming and has low efficiency, especially for large fields. Therefore, scientists and practitioners devote much effort to the automatization of this process. There are two complementary approaches to insect identification: (i) direct, in which the insect (ultimately the species) is determined, and (ii) indirect, in which the damage caused by the insects is monitored and forms the basis on which to formulate the information about insect infestation. A mini-review of both approaches is presented in this work. Additionally, the advantages and disadvantages of each are briefly described. Methods of insect identification are still characterized by relatively small selectivity and efficiency, therefore it is necessary to keep searching for new methods and improve the development of existing ones. The goal of such systems should be to work in real time and be inexpensive to run, enabling widespread use amongst farmers. A possible solution seems to be integrating various techniques (sensor fusion) into a single measurement system. © 2020 Society of Chemical Industry.

Purpose: The zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules. Methods: The study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides. Results: BacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide. Conclusion: All naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.

Helicobacter pylori represents a global health threat with around 50% of the world population infected. Due to the increasing number of antibiotic-resistant strains, new strategies for eradication of H. pylori are needed. In this study, we suggest purine nucleoside phosphorylase (PNP) as a possible new drug target, by characterising its interactions with 2- and/or 6-substituted purines as well as the effect of these compounds on bacterial growth. Inhibition constants are in the micromolar range, the lowest being that of 6-benzylthio-2-chloropurine. This compound also inhibits H. pylori 26695 growth at the lowest concentration. X-ray structures of the complexes of PNP with the investigated compounds allowed the identification of interactions of inhibitors in the enzyme’s base-binding site and the suggestion of structures that could bind to the enzyme more tightly. Our findings prove the potential of PNP inhibitors in the design of drugs against H. pylori.

nie dostarczono

The bioconjugate of alginate and curcumin (AA-CUR) was synthesized in a simple, one-step process and used to prepare the stable calcium cross-linked spherical micelles serving as a delivery vehicle for curcumin. Above its critical micelle concentration (0.6 mg/ml) AA-CUR forms colloidally stable micelles of ca. 200 nm. Prolonged, well controlled release of curcumin was observed from the AA-CUR micelles cross-linked with calcium ions for 5 h under the physiological conditions. To assess the safety of applying the bioconjugate into the bloodstream for possible anti-cancer applications, the interaction between AA-CUR and cells isolated from human blood was analyzed. No red cells aggregation or hemolysis was observed. AA-CUR was also shown to have no significant cytotoxicity to the human Peripheral Blood Mononuclear Cells (PBMC) isolated from peripheral blood of healthy donors and to the mouse primary brain endothelial cells. To evaluate the efficiency of the obtained bioconjugate in the anti-cancer therapy, the micellar solution of AA-CUR was tested against various cancer cells lines: mammary carcinoma 4T1, melanoma B16F10 and colon carcinoma CT26-CEA and MC38-CEA. The bioconjugate at the concentration of 0.7 mg/ml decreased the viability of cancer cells by ca. 80%. The cellular uptake of AA-CUR was rapid and the highest micelles accumulation was detected within 1 h after treatment. The AA-CUR micellar system can serve as an effective and safe delivery vehicle for curcumin.

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO2, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.

© 2021 Madej et al., Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface., This study was supported in part by grants from Spanish and Catalan public and private bodies (PID2019-107725RG-I00, 2017SGR3, and Fundació “La Marató de TV3” 201815 to F.X.G.-R.); National Science Centre, Poland grants UMO-2015/19/N/NZ1/ 00322 to M.M., UMO-2012/04/A/NZ1/00051 to J.P., and UMO-2016/23/N/NZ1/01513 to Z.N.; Polish Ministry of Science and Higher Education grant 1306/MOB/IV/2015/0 to M. K.; U.S. National Institutes of Health, NIDCR, grants DE09761 and DE026280 to J.P.; and Medical Research Council grant MR/P012175/2 to M.A.C.

Polymeric nanomaterials have become a prominent area of research in the field of drug delivery. Their application in nanomedicine can improve bioavailability, pharmacokinetics, and, therefore, the effectiveness of various therapeutics or contrast agents. There are many studies for developing new polymeric nanocarriers; however, their clinical application is somewhat limited. In this review, we present new complex and multifunctional polymeric nanocarriers as promising and innovative diagnostic or therapeutic systems. Their multifunctionality, resulting from the unique chemical and biological properties of the polymers used, ensures better delivery, and a controlled, sequential release of many different therapeutics to the diseased tissue. We present a brief introduction of the classical formulation techniques and describe examples of multifunctional nanocarriers, whose biological assessment has been carried out at least in vitro. Most of them, however, also underwent evaluation in vivo on animal models. Selected polymeric nanocarriers were grouped depending on their medical application: anti-cancer drug nanocarriers, nanomaterials delivering compounds for cancer immunotherapy or regenerative medicine, components of vaccines nanomaterials used for topical application, and lifestyle diseases, ie, diabetes.

The binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.

Camptothecin is a natural compound with excellent antitumor activity over a wide spectrum of human and animal cancers. However, the direct formulation is limited by insolubility in water and other biocompatible solvents, low stability in physiologic conditions, rapid plasma clearance and high systemic toxicity. In this study, pegylated polyelectrolyte nanocapsules were utilized to prepare camptothecin nanocarriers. Nanosize droplets containing the camptothecin were encapsulated in polyelectrolyte multilayer shells formed by sequential adsorption of polyelectrolytes technique. The nanocapsules were pegylated for passive targeting through the adsorption of the pegylated polyelectrolyte. The biological effects of encapsulated camptothecin on two tumor cell lines: mouse colon carcinoma cell line CT26-CEA and the mouse mammary carcinoma cell line 4T1 were studied. Obtained results suggest, that encapsulated drug, as well as free one, showed similar activity on the tested cells.

Alicja Karabasz,1 Krzysztof Szczepanowicz,2 Agnieszka Cierniak,1,3 Joanna Bereta,1 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, Poland; 3Department of Biochemistry, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Kraków University, Kraków, Poland Background: Toxicity of nanomaterials is one of the most important factors limiting their medical application. Evaluation of in vitro nanotoxicity allows for the identification and elimination of most of the toxic materials prior to animal testing. The current knowledge of the possible side effects of biodegradable nanomaterials, such as liposomes and polymeric organic nanoparticles, is limited. Previously, we developed a potential drug delivery system in the form of nanocapsules with polyelectrolyte, biodegradable shells consisting of poly-L-lysine and poly-L-glutamic acid (PGA), formed by the layer-by-layer adsorption technique. Methods: Hemolysis assay, viability tests, flow cytometry analysis of vascular cell adhesion molecule-1 expression on endothelium, analysis of nitric oxide production, measurement of intracellular reactive oxygen species levels, detection of antioxidant enzyme activity, and analysis of DNA damage with comet assay were performed to study the in vitro toxicity of nanocapsules. Results: In this work, we present the results of an in vitro analysis of toxicity of five-layer positively charged poly-L-lysine–terminated nanocapsules (NC5), six-layer negatively charged PGA-terminated nanocapsules (NC6) and five-layer PEGylated nanocapsules (NC5-PEG). PGA and polyethylene glycol (PEG) were used as two different “stealth” polymers. Of all the polyelectrolyte nanocapsules tested for blood compatibility, only cationic NC5 showed acute toxicity toward blood cells, expressed as hemolysis and aggregation. Neither NC6 nor NC5-PEG had proinflammatory activity evaluated through changes in the expression of NF-κB–dependent genes, iNOS and vascular cell adhesion molecule-1, induced oxidative stress, or promoted DNA damage in various cells. Conclusion: Our studies clearly indicate that PGA-coated (negatively charged) and PEGylated polyelectrolyte nanocapsules do not show in vitro toxicity, and their potential as a drug delivery system may be safely studied in vivo. Keywords: polyelectrolyte nanocapsules, layer-by-layer, nanotoxicity, oxidative stress, genotoxicity

The mechanism of green fluorescent protein (GFP) chromophore formation is still not clearly defined. Two mechanisms have been proposed: cyclisation-dehydration-oxidation (Mechanism A) and cyclisation-oxidation-dehydration (Mechanism B). To distinguish between these mechanisms, we generated a non-fluorescent mutant of GFP, S65T/G67A-GFP. This mutant folds to a stable, native-like structure but lacks fluorescence due to interruption of the chromophore maturation process. Mass spectrometric analysis of peptides derived from this mutant reveal that chromophore formation follows only mechanism A, but that the final oxidation reaction is suppressed. This result is unexpected within the pool of examined GFP mutants, since for the wild-type GFP, there is strong support for mechanism B.

In der Praxis erfolgen chemische Regulierungsmasnahmen der Feldmaus meist zu spat, wenn der Hohepunkt der Mauseplage schon erreicht ist. Hohe Umweltbelastungen, sowie hohe Kosten fur den Landwirt sind die Folgen. Hochaufgeloste Luftbilder von Drohnen (UAV) bieten eine Moglichkeit, Feldmauskolonien in einem Feld zu erkennen und deren Position uber GPS zu bestimmen. Eine Bekampfung von Feldmausen konnte dann rechtzeitig und zielgenau noch vor Beginn einer Massenvermehrung auf Feldebene erfolgen. Im Jahr 2015 wurden im Mai und Juni mit einer Drohne in einem heterogenen Winterweizenfeld mit Feldmausvorkommen Luftbildaufnahmen erstellt. An 20 Messpunkten mit unterschiedlicher Bestandsdichte erfolgten Handbonituren verschiedener Pflanzenparameter. Bereits zum ersten Termin waren die Auswirkungen des Trockenstresses zu beobachten. Mausenester ahneln aufgrund der abgefressenen Vegetation hinsichtlich der Farbung und der Helligkeit den Fahrspuren. Daher wurden die spektralen Signaturen der Fahrspuren fur deren Klassifizierung genutzt. Es ergaben sich Unterschiede im Lab-Farbraum zwischen den vegetationsfreien Fahrgassen und dem Pflanzenbestand, wobei sich diese auch innerhalb der zwei Klassen von Luftbild zu Luftbild anderten. Im ersten Luftbild konnten die funf vorhandenen Mausenester durch die angewendeten Bildverarbeitungsschritte richtig erkannt werden. In den darauffolgenden Bildern wurden durch die Trockenschaden nur noch vier Nester klassifiziert, wobei im letzten Bild eine grose Anzahl falsch positiv klassifizierter Nester auftrat.

Control of nonspecific/specific protein adsorption is the main goal in the design of novel biomaterials, implants, drug delivery systems, and sensors. The specific functionalization of biomaterials can be achieved by proper surface modification. One of the important strategies is covering the materials with functional coatings. Therefore, our work aimed to functionalize multilayer coating to control nonspecific/specific protein adsorption. The polyelectrolyte coating was formed using a layer-by-layer technique (LbL) with biocompatible polyelectrolytes poly-L-lysine hydrobromide (PLL) and poly-L-glutamic acid (PGA). Nonspecific protein adsorption was minimized/eliminated by pegylation of multilayer films, which was achieved by adsorption of pegylated polycations (PLL-g-PEG). The influence of poly (ethylene glycol) chain length on eliminating nonspecific protein adsorption was confirmed. Moreover, to achieve specific protein adsorption, the multilayer film was also functionalized by immobilization of antibodies via a streptavidin bridge. The functional coatings were tested, and the adsorption of the following proteins confirmed the ability to control nonspecific/specific adsorption: human serum albumin (HSA), fibrinogen (FIB), fetal bovine serum (FBS), carcinoembryonic antigen human (CEA) monitored by quartz crystal microbalance with dissipation (QCM-D). AFM imaging of unmodified and modified multilayer surfaces was also performed. Functional multilayer films are believed to have the potential as a novel platform for biotechnological applications, such as biosensors and nanocarriers for drug delivery systems.

The title compound is an excellent substrate for E. coli PNP, as well as for its D204N mutant. The main product of the synthetic reaction is N9-riboside, but some amount of N7-riboside is also present. Surprisingly, 1,N6-ethenoadenine is also ribosylated by both wild-type and mutated (N243D) forms of calf PNP, which catalyze the synthesis of a different riboside, tentatively identified as N6-β-D-ribosyl-1,N6-ethenoadenine. All ribosides are susceptible to phosphorolysis by the E. coli PNP (wild type). All the ribosides are fluorescent and can be utilized as analytical probes.

Despite progress in the development of novel pharmacological compounds, their efficacy in the treatment of neuropathologies is not satisfactory. One strategy to achieve safe and efficient brain targeting therapy is to design nanocarriers capable of transporting antipsychotic drugs through the BBB (without affecting the normal functions of the barrier) in a defined part of the brain. Here we investigate the interaction of clozapine-loaded polymeric Nano capsules (CLO-NCs) with hCMEC/D3 (human cerebral microvascular endothelial cells, D3 clone) cells that constitutes an in vitro model of the blood brain barrier (BBB). CLO-NCs (average size of 100nm) were constructed by the technique of sequential adsorption of polyelectrolytes (LbL), using biocompatible polyanion PGA (Poly-l-glutamic acid sodium salt) and polycation PLL (poly L-lysine) on clozapine-loaded nano-emulsion cores. Pegylated external layers were prepared using PGA-g(39)-PEG (PGA grafted by PEG poly(ethylene glycol)). The influence of the physicochemical properties of the CLO-NCs (charge, size, surface modification) on cell viability was determined. Advanced studies of CLO-NCs internalization (including endocytosis and transcytosis experiments) using confocal microscopy, flow cytometry and fluorescence spectroscopy are presented. Our results indicate that among the studied NCs, the pegylated clozapine-loaded NCs were the most protected from their uptake by macrophages, and they were the least toxic to hCMEC/D3 cells. They were also the most efficient in the transcytosis experiment, which serves as an indicator of their ability to cross a model BBB.

Background Achieving sustainability of agricultural production is one of the key challenges for humanity. Minimizing the use of chemicals and replacing them with biological plant protection is, firstly, fully in line with the agriculture's development strategy and, secondly, confirmed to be achievable in practice. For instance, many researchers point to the potential of using Trichogramma spp. to control Ostrinia nubilalis in maize. However, the application methods for preparations containing Trichogramma spp. are still at the stage of development. So far, ground application proved time and energy consuming. Aerial application using ultralight aircraft is a new option. Results The present study presents the results of tests on the effectiveness of applying Trichogramma spp. from the board of an ultralight gyroplane. A 6-year experiment was conducted on a maize monoculture located in south-western Poland. The results indicate that the low-height aerial application allows precise dosing and satisfactory distribution of the biopesticide. The efficacy of 60-85% (depending on the year) of the gyroplane-based spraying operations was comparable with that observed for ground application. Conclusion Considering the speed and high efficiency of the treatment, using a gyroplane as a carrier of the biopesticide application system is a good alternative to the other methods. © 2020 Society of Chemical Industry.

Here, we designed a novel Gadolinium (Gd) labeled drug-loaded polyelectrolyte nanocarriers for theranostics. The nanocarriers were formed via layer-by-layer technique with biodegradable polyelectrolytes: PLL (Poly-L-lysine), PLL-Gd (Gadolinium-labeled Poly-L-lysine) and PGA (Poly-L-glutamic acid). Anticancer drug (Paclitaxel) was encapsulated in the formed nanocarriers. The average size of synthesized nanocarriers was around 150 nm. The empty gadolinium labeled nanocarriers did not show any deleterious effects on tested cells (CT26-CEA, B16F10, 4T1 and PBMC), whereas encapsulated paclitaxel retained its cytotoxic/cytostatic activity. Using T2 and T1 NMR relaxation measurements with 9.4 T preclinical MRI scanner, we demonstrated that gadolinium labeled nanocarriers can be detected due to a locally altered contrast in the MR image. Thus, they may become a promising platform for future theranostic applications.

Background Curcumin is a natural polyphenol with anti-inflammatory, chemopreventive and anticancer activity. However, its high hydrophobicity and poor bioavailability limit its medical application. The development of nanocarriers for curcumin delivery is an attractive approach to overcome its low bioavailability and fast metabolism in the liver. We synthesized a blood compatible alginate-curcumin conjugate, AA-Cur, which formed colloidally stable micelles of approximately 200 nm and, as previously shown, exerted strong cytotoxicity against mouse cancer cell lines. Here we analyze in vivo toxicity and antitumor activity of AA-Cur in two different mouse tumor models. Method Potential toxicity of intravenously injected AA-Cur was evaluated by: i) analyses of blood parameters (morphology and biochemistry), ii) histology, iii) DNA integrity (comet assay), and iv) cytokine profiling (flow cytometry). Antitumor activity of AA-Cur was evaluated by measuring the growth of subcutaneously inoculated colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in control mice vs mice treated with AA-Cur. Results Injections of four doses of AA-Cur did not reveal any toxicity of the conjugate, thus indicating the safety of its use. AA-Cur elicited moderate anti-tumor activity toward colon MC38-CEA or breast 4T1 carcinomas. Conclusion The tested conjugate of alginate and curcumin, AA-Cur, is non-toxic and safe, but exhibits limited anticancer activity.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrAHp) belongs to the well conserved family of serine proteases. HtrAHp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrAHp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrAHp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrAHp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrAHp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrAHp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrAHp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

Replacing water with dimethyl sulfoxide (DMSO) completely reshapes the free-energy landscapes of solvated proteins. In DMSO, a powerful hydrogen-bond (HB) acceptor, formation of HBs between backbone NH groups and solvent is favored over HBs involving protein's carbonyl groups. This entails a profound structural disruption of globular proteins and proteinaceous aggregates (e.g., amyloid fibrils) upon transfer to DMSO. Here, we investigate an unusual DMSO-induced conformational transition of β

Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation.

The title compound is an excellent substrate for E. coli PNP, as well as for its D204N mutant. The main product of the synthetic reaction is N9-riboside, but some amount of N7-riboside is also present. Surprisingly, 1,N

IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo.

Despite the limitations in the use of aerial applications resulting from Directive 2009/128/EC of the European Parliament and of the Council and the Plant Protection Agents Act (OJ L item 455. 2013), scientific progress in engineering enables the development of new, environmentally safe technologies to expand the use of agroaviation. This paper proposes an innovative method of biological protection of corn against Ostrinia nubilalis with the use of an autogyro and presents the results of these operations. An autogyro adaptation for forest applications is proposed, and the preliminary results of a spray uniformity assessment are presented. Based on a two-year study (542.5 ha), the introduction of the Tricholet preparation against Ostrinia nubilalis was found to be highly effective (73.55%), which is a positive indication for the innovative autogyro method for introducing Trichogramma evanescens. Similarly, positive results were obtained for a liquid agent application using an autogyro-mounted installation: an even coverage of the area was achieved across the entire spray path width while maintaining the required dosing of the plant protection agent.

For a number of enzymes composed of several subunits with the same amino acid sequence, it was documented, or suggested, that binding of a ligand, or catalysis, is carried out by a single subunit. This phenomenon may be the result of a pre-existent asymmetry of subunits or a limiting case of the negative cooperativity, and is sometimes called "half-of-the-sites binding (or reactivity)" for dimers and could be called "part-of-the-sites binding (or reactivity)" for higher oligomers. In this article, we discuss molecular mechanisms that may result in "part-of-the-sites binding (and reactivity)", offer possible explanations why it may have a beneficial role in enzyme function, and point to experimental problems in documenting this behaviour. We describe some cases, for which such a mechanism was first reported and later disproved. We also give several examples of enzymes, for which this mechanism seems to be well documented, and profitable. A majority of enzymes identified in this study as half-of-the-sites binding (or reactive) use it in the flip-flop version, in which "half-of-the-sites" refers to a particular moment in time. In general, the various variants of the mechanism seems to be employed often by oligomeric enzymes for allosteric regulation to enhance the efficiency of enzymatic reactions in many key metabolic pathways.

Background Purine-nucleoside phosphorylase (PNP) is known as a tool for the synthesis of various nucleosides and nucleoside analogues. Mechanism, properties, molecular diversity and inhibitors of PNP, particularly these of pharmacological significance, are briefly characterized. Methods UV and fluorescence spectroscopy was used for kinetic experiments, and HPLC chromatography for product analyses. Results Applications of various forms of PNP to synthesis of selected fluorescent nucleosides, particularly ribosides of 1,N6-ethenoadenine and various 8-azapurines (triazolo[4,5-d]pyrimidines) are reviewed. Different specificity of various PNP forms is described towards nucleobase and analogue substrates as well as variable ribosylation sites observed in some reactions, with a possibility to further modify these features via the site-directed mutagenesis. Conclusion Present and future applications of the fluorescent or fluorogenic ribosides are discussed, with particular emphasis on biochemical and clinical analyses with improved sensitivity.

Homotrimeric mammalian purine nucleoside phosphorylase (PNP) plays a key role in the nucleoside and nucleotide metabolic salvage pathway. Each monomer in the active PNP trimer is composed of a central β-sheet flanked by several α-helices. We investigated the stability of calf PNP using analytical ultracentrifugation, differential scanning calorimetry, circular dichroism, and UV absorption spectroscopy. The results demonstrate that the activity decline (due to protein aging after isolation from cells) of wild type PNP and its two mutants with point mutations in the region of monomer-monomer interface, is accompanied by a decrease of the population of the trimeric enzyme and an increase of the population of its aggregated forms. The data do not indicate a significant population of either folded or unfolded PNP monomers. The enzyme with specific activity lower than the maximal shows a decrease of the helical structure, which can make it prone to aggregation. The presence of phosphate stabilizes the enzyme but leads to a more pronounced aggregation above the melting temperature. These results suggest that the biological role of packing of the PNP monomers into a trimeric structure is to provide the stability of the enzyme since the monomers are not stable in solution.

Although many enzymes are homooligomers composed of tightly bound subunits, it is often the case that smaller assemblies of such subunits, or even individual monomers, seem to have all the structural features necessary to independently conduct catalysis. In this study, we investigated the reasons justifying the necessity for the hexameric form of Escherichia coli purine nucleoside phosphorylase – a homohexamer composed of three linked dimers – since it appears that the dimer is the smallest unit capable of catalyzing the reaction, according to the currently accepted mechanism. Molecular modelling was employed to probe mutations at the dimer–dimer interface that would result in a dimeric enzyme form. In this way, both in silico and in vitro, the hexamer was successfully transformed into dimers. However, modelling and solution studies show that, when isolated, dimers cannot maintain the appropriate three-dimensional structure, including the geometry of the active site and the position of the catalytically important amino acids. Analytical ultracentrifugation proves that E. coli purine nucleoside phosphorylase dimeric mutants tend to dissociate into monomers with dissociation constants of 20–80 μm. Consistently, the catalytic activity of these mutants is negligible, at least 6 orders of magnitude smaller than for the wild-type enzyme. We conclude that the hexameric architecture of E. coli purine nucleoside phosphorylase is necessary to provide stabilization of the proper three-dimensional structure of the dimeric assembly, and therefore this enzyme is the obligate (obligatory) hexamer. Structured digital abstract ●PNP and PNP bind by molecular sieving (1, 2, 3, 4)

Despite high similarity of canine respiratory coronavirus (CRCoV), bovine coronavirus, (BCoV) and human coronavirus OC43 (HCoV-OC43), these viruses differ in species specificity. For years it was believed that they share receptor specificity, utilizing sialic acids for cell surface attachment, internalization, and entry. Interestingly, careful literature analysis shows that viruses indeed bind to the cell surface via sialic acids, but there is no solid data that these moieties mediate virus entry. In our study, using a number of techniques, we showed that all three viruses are indeed able to bind to sialic acids to a different extent, but these molecules render the cells permissive only for the clinical strain of HCoV-OC43, while for others they serve only as attachment receptors. CRCoV and BCoV appear to employ human leukocyte antigen class I (HLA-1) as the entry receptor. Furthermore, we identified heparan sulfate as an alternative attachment factor, but this may be related to the cell culture adaptation, as in ex vivo conditions, it does not seem to play a significant role. Summarizing, we delineated early events during CRCoV, BCoV, and HCoV-OC43 entry and systematically studied the attachment and entry receptor utilized by these viruses.

The enhanced green fluorescent protein (EGFP) is broadly used as an intracellular fluorescent marker. Its numerous applications require a comprehensive understanding of EGFP folding properties, as well as the properties of EGFP-derived mutants. Herein, we report on the spectroscopic characterization of two single-cysteine mutants, C48S-EGFP and C70S-EGFP, which were prepared to monitor the protein folding dynamics by EPR, specifically in the close vicinity of both cysteine residues. Chromophore absorption, excitation and emission spectra for these mutants were similar to those for EGFP. Both single- cysteine mutants were more susceptible to aggregation than EGFP in E. coli. C48S-EGFP exhibited similar resistance to chem- ical denaturation as EGFP. In contrast, C70S-EGFP was less resistant to denaturation in guanidine hydrochloride and its folding was less efficient. Hence, C48S-EGFP seems to be a more suitable probe for EPR measurements than C70S-EGFP.

Sulphate mineralization precipitated around a geyser located above the village of Pinchollo, Chivay district and below Hualca Hualca volcano (6025 m a.s.l.) in the Western Cordillera of southern Peru is described. The geyser is one of many manifestations of thermal activity in the Arequipa department. Its age is estimated to be Upper Pleistocene–Holocene, as the discharge point lies at the intersection of a fault system with latitudinal dip-slip fault cutting a volcanic-debris avalanche of probably Pleistocene age. Thermal waters present in the Chivay district are mainly chloride-rich with a neutral pH. They are rich in Li, Sr, and B. The water erupting in the geyser boils at about 85 °C, as it lies at some 4353 m a.s.l. The minerals examined, of various habits and various yellow, orange and white colours were precipitated on the soil and on plants close to the geyser (location 1), on the walls of a 1 m diameter pothole filled with boiling water (location 1a) and at a distance of some 100 m to the west of the geyser (location 2). All are sulphates. Their chemical composition is fairly simple, consisting of Al, Fe, K, Mg, Ca, S, NH 4 and O, and all display chemical zoning. But the phase composition is more complex. In all locations, alunogene, copiapite, coquimbite, tschermigite and gypsum are present. Close to the geyser (location 1) magnesium-containing sulphates, namely, boussingaultite and pickeringite also occur. Iron sulphates such as mohrite and rozenite precipitate on the walls of the pothole (location 1a). Sulphates containing potassium such as jarosite, alunite and voltaite–voltaite (Mg) dominate among the efflorescences in location 2, where hematite was also noted. Any quartz and kaolinite or illite/mica admixture identified in some samples derives from adjacent soil. The present geothermal system does not involve the deposition of precious-metal deposits such as those associated with an earlier deep-going epithermal system that scavenged a large volume of rock. Most likely, as the present-day thermal waters do not involve a juvenile-water component, the geyser waters derive from a shallower source.

Microaerophilic bacterium Helicobacer pylori is a well known human pathogen involved in the development of many diseases. Due to the evergrowing infection rate and increase of H. pylori antibiotic resistence, it is of utmost importance to find a new way to attack and eradicate H. pylori. The purine metabolism in H. pylori is solely dependant on the salvage pathway and one of the key enzymes in this pathway is purine nucleoside phosphorylase (PNP). In this timely context, we report here the basic biochemical and structural characterization of recombinant PNP from the H. pylori clinical isolate expressed in Escherichia coli. Structure of H. pylori PNP is typical for high molecular mass PNPs. However, its activity towards adenosine is very low, thus resembling more that of low molecular mass PNPs. Understanding the molecular mechanism of this key enzyme may lead to the development of new drug strategies and help in the eradication of H. pylori.

The results of several decades of studying the catalytic mechanism of Escherichia coli purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared. The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme. (doi: 10.5562/cca2116)

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.

It is widely accepted that oxidized low-density lipoproteins and local infections or endotoxins in circulation contribute to chronic inflammatory process at all stages of atherosclerosis. The hallmark cells of atherosclerotic lesions—monocytes and macrophages—are able to detect and integrate complex signals derived from lipoproteins and pathogens, and respond with a spectrum of immunoregulatory cytokines. In this study, we show strong inhibitory effect of oxLDLs on anti-inflammatory interleukin-10 production by monocytes responding to TLR2 and TLR4 ligands. In contrast, pro-inflammatory tumor necrosis factor secretion was even slightly increased, when stimulated with lipopolysaccharide from Porphyromonas gingivalis—an oral pathogen associated with atherosclerosis. The oxLDLs modulatory activity may be explained by altered recognition of pathogen-associated molecular patterns, which involves serum proteins, particularly vitronectin. We also suggest an interaction between vitronectin receptor, CD11b, and TLR2. The presented data support a novel pathway for pathogen-accelerated atherosclerosis, which relies on oxidized low-density lipoprotein-mediated modulation of anti-inflammatory response to TLR ligands. Electronic supplementary material The online version of this article (doi:10.1007/s10753-012-9472-3) contains supplementary material, which is available to authorized users.

The catalytic mechanism of Escherichia coli purine nucleoside phosphorylase (PNP) is revised using site-directed mutagenesis, kinetic studies and structure determinations. The experimental evidence on the role of the particular catalytic amino acid during catalysis has not been available. Therefore, the active site mutants Arg24Ala, Asp204Ala, Asp204Asn, Arg217Ala and Asp204Ala/Arg217Ala were prepared and their kinetics and thermodynamic studies were carried out. The activity tests with natural substrates and 7-methylguanosine confirmed the earlier hypothesis, that catalysis involves protonation of the purine base at position N7 by Asp204, which is triggered by Arg217. The crystal structures of the wild type in complexes with phosphate and sulphate, respectively, and of the Arg24Ala mutant in complex with phosphate/sulphate were determined. The structural data show that previously observed conformational change is a result of the phosphate binding and its interaction with Arg24. As E. coli PNP is a promising candidate for the tumour-directed gene therapy, our results may also help to design efficient mutants useful in gene therapy.

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.

9-(5′,5′-Difluoro-5′-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5′,5′-difluoro-5′-phosphonopentyl)guanine (DFPP-G) with calf-spleen PNP. DFPP-DG and its analogous compounds were synthesized by the Sonogashira coupling reaction between a 9-deaza-9-iodoguanine derivative and ω-alkynyldifluoromethylene phosphonates as a key reaction. The experimental details focused on the synthetic chemistry along with some insights into the physical and biological properties of newly synthesized DFPP-DG derivatives are disclosed.