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Titanium oxide nanoparticles modified with D-(+)-mannose were obtained. In the process of their formation, they were conjugated with an active substance (tadalafil). The physicochemical properties of the obtained products were assessed, and the size and electrokinetic potential were determined using a dynamic light scattering technique. X-ray diffractometry was applied in order to define the crystalline properties, and Fourier-transform infrared spectroscopy was used to confirm the formation of the desired products. It was possible to obtain TiO2 coated with D-(+)-mannose. The average size of nanoparticles was between 230 and 268 nm. The release of the active substance from the product over a time period of three hours was assessed against the reference material, which was not modified by D-(+)-mannose. The results indicate that covering titanium oxide nanoparticles with the modifying substance favours a slower rate of release for the active substance, which is the desired effect from a pharmacological point of view. The releasing of active substance from modified products was even 68% slower than that from the reference product. These modified titanium oxides are promising materials that may have found an application as drug carriers. Graphic Abstract

[EN] Cancer is one of the deadliest illness globally. Searching for new solutions in cancer treatments is essential because commonly used mixed, targeted and personalized therapies are sometimes not sufficient or are too expensive for common patients. Sugar fatty acid esters (SFAEs) are already well-known as promising candidates for an alternative medical tool. The manuscript brings the reader closer to methods of obtaining various SFAEs using combined biological, chemical and enzymatic methods. It presents how modification of SFAE's hydrophobic chains can influence their cytotoxicity against human skin melanoma and prostate cancer cell lines. The compound's cytotoxicity was determined by an MTT assay, which followed an assessment of SFAEs' potential metastatic properties in concentrations below IC50 values. Despite relatively high IC50 values (63.3-1737.6 μM) of the newly synthesized SFAE, they can compete with other sugar esters already described in the literature. The chosen bioactives caused low polymerization of microtubules and the depolymerization of actin filaments in nontoxic levels, which suggest an apoptotic rather than metastatic process. Altogether, cancer cells showed no propensity for metastasis after treating them with SFAE. They confirmed that lactose-based compounds seem the most promising surfactants among tested sugar esters. This manuscript creates a benchmark for creation of novel anticancer agents based on 3-hydroxylated fatty acids of bacterial origin., Wojciech Snoch acknowledges the support of InterDokMed project no. POWR.03.02.00-00-I013/16. We greatly acknowledge the joint consortium “Interdisciplinary Centre of Physical, Chemical and Biological Sciences” of ICSC PAS and INP PAS for providing access to the Agilent 1290 Infinity System with an automatic autosampler and an MS Agilent 6460 Triple Quad Detector.

Subnuclear localization of topoisomerase I (top I) is determined by its DNA relaxation activity and a net of its interactions with in majority unidentified nucleolar and nucleoplasmic elements. Here, we recognized SR protein SRSF1 (Serine/arginine-rich splicing factor 1, previously known as SF2/ASF) as a new element of the net. In HeLa cells, overexpression of SRSF1 recruited top I to the nucleoplasm whereas its silencing concentrated it in the nucleolus. Effect of SRSF1 was independent of top I relaxation activity and was the best pronounced for the mutant inactive in relaxation reaction. In HCT116 cells where top I was not released from the nucleolus upon halting relaxation activity, it was also not relocated by elevated level of SRSF1. Out of remaining SR proteins, SRSF5, SRSF7, and SRSF9 did not influence the localization of top I in HeLa cells whereas overexpression of SRSF2, SRSF3, SRSF6, and partly SRSF4 concentrated top I in the nucleolus, most possibly due to the reduction of the SRSF1 accessibility. Specific effect of SRSF1 was exerted because of its distinct RS domain. Silencing of SRSF1 compensated the deletion of the top I N-terminal region, individually responsible for nucleoplasmic localization of the mutant, and restored the wild-type phenotype of deletion mutant localization. SRSF1 was essential for the camptothecin-induced clearance from the nucleolus. These results suggest a possible role of SRSF1 in establishing partition of top I between the nucleolus and the nucleoplasm in some cell types with distinct combinations of SR proteins levels.

The gastrointestinal hydrolysis of food proteins has been portrayed in scientific literature to predominantly depend on the activity and specificity of proteolytic enzymes. Human bile has not been considered to facilitate proteolysis in the small intestine, but rather to assist in intestinal lipolysis. However, human bile can potentially influence proteins that are largely resistant to gastric digestion, and which are mainly hydrolysed after they have been transferred to the small intestine. We used purified and food-grade bovine milk β-lactoglobulin (βLg) to assess the impact of bile salts (BS) on the in vitro gastrointestinal digestion of this protein. Quantitative analysis showed that the proteolysis rate increased significantly with increasing BS concentration. The effect was consistent regardless of whether individual BS or real human bile samples, varying in BS concentrations, were used. The total BS content of bile was more important than its BS composition in facilitating the proteolysis of βlg. We also show that the impact of human bile observed during the digestion of purified βLg and βLg-rich whey protein isolate can be closely replicated by the use of individual BS mixed with phosphatidylcholine. This could validate simple BS/phosphatidylcholine mixtures as human-relevant substitutes of difficult-to-obtain human bile for in vitro proteolysis studies.

In this study, we evaluate the factors which determine the reactivity of divalent metal ions in the spontaneous formation of metallochlorophylls, using experimental and computational approaches. Kinetic studies were carried out using pheophytin a in reactions with various divalent metal ions combined with non- or weakly-coordinative counter ions in a series of organic solvents. To obtain detailed insights into the solvent effect, the metalations with the whole set of cations were investigated in three solvents and with Zn2+ in seven solvents. The reactions were monitored using electronic absorption spectroscopy and the stopped-flow technique. DFT calculations were employed to shed light on the role of solvent in activating the metal ions towards porphyrinoids. This experimental and computational analysis gives detailed information regarding how the solvent and the counter ion assist/hinder the metalation reaction as activators/inhibitors. The metalation course is dictated to a large extent by the reaction medium, via either the activation or deactivation of the incoming metal ion. The solvent may affect the metalation in several ways, mainly via H-bonding with pyrrolenine nitrogens and the activation/deactivation of the incoming cation. It also seems to affect the activation enthalpy by causing slight conformational changes in the macrocyclic ligand. These new mechanistic insights contribute to a better understanding of the “metal–counterion–solvent” interplay in the metalation of porphyrinoids. In addition, they are highly relevant to the mechanisms of metalation reactions catalyzed by chelatases and explain the differences between the insertion of Mg2+ and other divalent cations. Electronic supplementary material The online version of this article (doi:10.1007/s00775-017-1472-1) contains supplementary material, which is available to authorized users.

Human topoisomerase I is partitioned between the nucleolus and the nucleoplasm in the interphase cells. Under unstressed conditions it is concentrated in the first compartment but nucleolar concentration of the full length protein is lost after inactivation of relaxation activity. Due to the above, subnuclear localization of topoisomerase I is linked with DNA relaxation activity of topoisomerase I. Looking for other factors responsible for subnuclear distribution of topoisomerase I, we studied here localization of the fluorescently tagged fragments and point mutants of topoisomerase I in HeLa cells. We found that two regions of topoisomerase I, the N-terminal and the linker domains, were critical for subnuclear localization of the enzyme. The linker domain and the distal region of the N-terminal domain directed topoisomerase I to the nucleolus, whereas the remaining region of the N-terminal domain was responsible for the nucleoplasmic localization. The effects exhibited by the regions which contributed to nuclear distribution of topoisomerase I were independent of DNA relaxation activity. Localization mutations in both domains complemented one another giving the wild-type phenotype for the double mutant. These results suggest a two-stage model of regulation of partitioning of topoisomerase I between the nucleolus and the nucleoplasm. The first stage is a net of interactions provided by the N-terminal and the linker domains. The other stage, accessible only if the first net is balanced, is driven by DNA relaxation activity. J. Cell. Biochem. 118: 407-419, 2017. © 2016 Wiley Periodicals, Inc.

Selol, an organic selenitetrigliceride formulation containing selenium at +4 oxidation level, has been suggested as anticancer drug. One of the causes of several diseases including cancer may be inflammation. This study aimed at determining the activity of Selol via measuring its effect on reactive oxygen species (ROS) generation, nuclear factor kappa B (NF-κB) activation, intercellular cell adhesion molecules-1 (ICAM-1), vascular cell adhesive molecule-1 (VCAM-1), and plateled-endothelial cell adhesive molecule-1 (PECAM-1) levels on control and on tumor necrosis factor-α (TNF-α)-stimulated human microvascular endothelial cells (HMEC-1). Cells were treated either with Selol 5% (4 or 8 μgSe/mL) or TNF-α (10 ng/mL) alone or with Selol concomitant with TNF-α. Selol treatment resulted in ROS generation, activation of NF-κB, downregulation of PECAM-1, VCAM-1 and slight upregulation ICAM-1 expression on the cell surface. TNF-α treatment reflected in sharp NF-κB activation, upregulation of both ICAM-1 and VCAM-1 in parallel with the downregulation of PECAM-1 expression on cell surface. Exposure to both compounds upregulated ICAM-1 and VCAM-1, downregulated PECAM-1 level on cell surface in parallel with no changes in level of NF-κB activation as compared with effects mediated by TNF-α alone. These results points to new look at Selol action since it shows a pro-inflammatory activity in parallel with effects on CAMs expression on the cell surface of human microvascular endothelial cells. However, since Selol enhances CAMs expression level when is present concomitantly with TNF-α this fact might suggest that selenium present in the condition of inflammation will make it worse.

Numerous formulations derived from the shiitake medicinal mushroom, Lentinus edodes, demonstrate anticancer activities. We hypothesized that isolates from selenium (Se)-enriched mycelia of L. edodes would possess stronger cancer-preventive properties than current preparations. The aim of this study was to investigate whether the presence of Se-methyl-seleno-L-cysteine in mycelial extracts of L. edodes affects their cytotoxic activity (makes them stronger) or whether they are as effective as Se-containing polysaccharides. Extracts were prepared from Se-containing mycelia under various conditions and assayed for cytotoxic activity in cancer (PC3 and HeLa) and normal (HMEC-1) cell lines. The chemical composition of the extracts was examined; specifically, the amounts of potentially cytotoxic Se compounds (methylselenocysteine, selenomethionine, and Se-containing polysaccharides) were measured. The relationship between extract composition and biological activity was characterized. Mycelial cultures were cultivated in a 10-L bioreactor in medium enriched with sodium selenite. Mycelial extracts were prepared either at 100°C or at 4°C in acidic solution. Total Se content was determined using the atomic absorption spectrometry method, and methylselenocysteine and selenomethionine contents were measured using reverse-phase high-performance liquid chromatography. Protein, carbohydrate, and polyphenolic contents were determined with spectrophotometric methods, and Se-containing polysaccharides were measured with the use of precipitation. Anticancer activity of mycelial extracts was examined using the MTT cell viability assay. Extracts containing Se-methyl-seleno-L-cysteine or Se-polysaccharides prepared at 4°C and 100°C, respectively, display moderate, time-dependent, specific cytotoxic activity in HeLa and PC3 cell lines. The effect in HeLa cells is more pronounced in the extract prepared at 4°C than at 100°C. The effect is almost equal for the PC3 cell line. However, both extracts have no effect or only slightly stimulate normal (HMEC-1) cell viability. The selective cytotoxic activity of L. edodes extracts in cancer (PC3 and HeLa) cells is due to the presence of both Se-methyl-seleno-L-cysteine and selenated polysaccharides, perhaps in combination with other active ingredients.

Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 µg/mL or 2 µg/mL) (0.2 µM or 0.8 µM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action.

Thiram (TMTD) is a widely used dithiocarbamate pesticide and fungicide and is one of potent contact allergens. In the light of known properties, thiram is also considered to be used as an inhibitor of inflammation. To investigate whether known pro-oxidative properties of thiram might be involved in immunogenic mechanisms, we carried out an in vitro study aimed at analysis of reactive oxygen species (ROS) generation, activation of NF-κB, expression of iNOS and COX-2, production of NO, PGE2 and IL-1β in murine macrophage cells (RAW 264.7). The cells were treated by thiram alone (0.5 µg/mL; 2 μM and 2 µg/mL; 8 μM) or concomitantly with bacterial endotoxin (LPS; 1 μg/mL). LPS was used as an endotoxin that triggers changes characteristic for inflammatory state of the cell. TMTD increased ROS production, level of oxidized glutathione (GSSG) and activated NF-κB. The consequence of NF-κB activation was the increase of IL-1β and NO production characteristic for inflammation. However, we did not observe changes in PGE2 concentration. We observed expression of iNOS, COX-2 proteins and NO and PGE2 production in macrophages treated with thiram concomitantly with LPS lower than those in cells stimulated with LPS alone. Thiram (2 µg/mL) decreased NF-κB activation and production of LPS-induced IL-1β. In conclusion, we demonstrated changes induced by TMTD characteristic for inflammation. Hence, it can be supposed that they may participate in the elicitation phase of allergic contact dermatitis induced by thiram. However, when TMTD acts concomitantly with LPS, it decreases the intensity of inflammation state in RAW 264.7.

The maintenance of copper homeostasis is critical for all cells. As learned from mice with disturbed copper metabolism, this trace element is also important for spermatogenesis. The experiments conducted in yeasts have demonstrated that appropriate copper level must be preserved to enable meiosis progression; however, increased copper level is toxic for cells. This study aims to analyze the expression profile of Atp7a and Atp7b and other genes encoding copper-related proteins during spermatogenesis in mice. Using the transcripts and protein detection techniques, we demonstrate that within seminiferous tubuli, ATP7A is mainly present in early meiotic germ cells (leptotene to pachytene spermatocytes) and in Sertoli cells (SCs). During spermatogenesis, the progression Atp7a expression profile corresponds to Slc31a1 (encoding copper importer CTR1) and Atox1 (encoding chaperon protein, which delivers copper from CTR1 to ATP7A and ATP7B) expression, suggesting that male germ cells retrieve copper and ATP7A protects them from copper overdose. In contrast, ATP7B protein is observed in SCs and near elongated spermatids; thus, its function seems to be related to copper extraction during spermiogenesis. This is the first study to give a comprehensive view on the activity of copper-related genes during spermatogenesis in mice.

Iron regulatory protein 1 (IRP1) is a cytosolic bifunctional [4Fe-4S] protein which exhibits aconitase activity or binds iron responsive elements (IREs) in untranslated regions of specific mRNA encoding proteins involved in cellular iron metabolism. Superoxide radical (O2.-) converts IRP1 from a [4Fe-4S] aconitase to a [3Fe-4S] "null" form possessing neither aconitase nor trans-regulatory activity. Genetic ablation of superoxide dismutase 1 (SOD1), an antioxidant enzyme that acts to reduce O2.- concentration, revealed a new O2.--dependent regulation of IRP1 leading to the reduction of IRP1 protein level and in consequence to the diminution of IRP1 enzymatic and IRE-binding activities. Here, we attempted to establish whether developmental changes in SOD1 activity occurring in the mouse liver, impact IRP1 expression. We show no correlation between hepatic SOD1 activity and IRP1 protein level neither in pre- nor postnatal period probably because the magnitude of developmental fluctuations in SOD1 activity is relatively small. The comparison of SOD1 activity in regards to IRP1 protein level in the liver of threeSOD1 genotypes (Sod1+/+, Sod1+/- and Sod1-/-) demonstrates that only drastic SOD1 deficiency leads to the reduction of IRP1 protein level. Importantly, we found that in the liver of fetuses lacking SOD1, IRP1 is not down-regulated. To investigate O2.--dependent regulation of IRP1 in a cellular model, we exposed murine RAW 264.7 and bone marrow-derived macrophages to paraquat, widely used as a redox cycler to stimulate O2.-production in cells. We showed that IRP1 protein level as well as aconitase and IRE-binding activities are strongly reduced in macrophages treated with paraquat. The analysis of the expression of IRP1-target genes revealed the increase in L-ferritin protein level resulting from the enhanced transcriptional regulation of the LFt gene and diminished translational repression of L-ferritin mRNA by IRP1. We propose that O2.--dependent up-regulation of this cellular protectant in paraquat-treated macrophages may counterbalance iron-related toxic effects of O2.-.

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O2-independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[2H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH.

A series of 5-aryl-1-alkylimidazole derivatives was synthesized using the van Leusen multicomponent reaction. The chemotype is the first example of low-basicity scaffolds exhibiting high affinity for 5-HT7 receptor together with agonist function. The chosen lead compounds 3-(1-ethyl-1H-imidazol-5-yl)-5-iodo-1H-indole (AGH-107, 1o, Ki 5-HT7 = 6 nM, EC50 = 19 nM, 176-fold selectivity over 5-HT1AR) and 1e (5-methoxy analogue, Ki 5-HT7 = 30 nM, EC50 = 60 nM) exhibited high selectivity over related CNS targets, high metabolic stability and low toxicity in HEK-293 and HepG2 cell cultures. A rapid absorption to the blood, high blood-brain barrier permeation and a very high peak concentration in the brain (Cmax = 2723 ng/g) were found for 1o after i.p. (5 mg/kg) administration in mice. The compound was found active in novel object recognition test in mice, at 0.5, 1 and 5 mg/kg. Docking to 5-HT7R homology models indicated a plausible binding mode which explain the unusually high selectivity over the related CNS targets. Halogen bond formation between the most potent derivatives and the receptor is consistent with both the docking results and SAR. 5-Chlorine, bromine and iodine substitution resulted in a 13, 27 and 89-fold increase in binding affinities, respectively, and in enhanced 5-HT1AR selectivity.

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.

Sodium diethyldithiocarbamate (DETC) is the main metabolite of disulfiram. Recently, we reported that mechanism of disulfiram cytotoxicity in V79 cells might be partially connected with thiol redox-state imbalance. Here, we examined the effect of DETC on the level of intracellular glutathione (GSH), protein oxidation (measured as PC—protein carbonyl content), lipid peroxidation (measured as TBARS—thiobarbituric acid reactive substances), antioxidant enzymatic defense, as well as on apoptosis. We used V79 Chinese hamster fibroblasts cells with and without modulated glutathione (GSH) level by N-acetyl-l-cysteine (NAC). We showed that treatment with DETC at concentrations that cause a moderate increase in thiol-state imbalance but not cell death stimulates oxidative stress measured as increased level of PC and TBARS, adaptive response of GSH-related enzymes and apoptosis. Our results show that cellular effects of DETC are partially attributable to the initial redox cellular state, since the increase of GSH level by NAC pre-treatment prevented the observed changes.

Dithiocarbamate fungicide zineb leads to disturbances in many cellular processes in Chinese hamster fibroblasts (V79 cells). We observed high apoptotic activity and oxidative stress induced in vitro by exposure of V79 cells to zineb demonstrated by statistically significant increase in lipid peroxidation (measured as TBARS – thiobarbituric acid reactive substances) as well as disturbances in glutathione homeostasis. Zineb also inhibited activity of topoisomerase I. Zineb did not show any effect on protein oxidation (measured by protein carbonyls content). N-acetyl- l -cysteine (NAC) suppressed cellular changes induced by this fungicide in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells induced by zineb. These results show that zineb can disturb many cellular processes via oxidative damage.

Peptide antibiotics are produced by a wide range of microorganisms. Most of them target the cell envelope, often by inhibiting cell wall synthesis. One of the resistance mechanisms against antimicrobial peptides is a detoxification module consisting of a two-component system and an ABC transporter. Upon the detection of such a compound, the two-component system induces the expression of the ABC transporter, which in turn removes the antibiotic from its site of action, mediating the resistance of the cell. Three such peptide antibiotic-sensing and detoxification modules are present in Bacillus subtilis . Here we show that each of these modules responds to a number of peptides and confers resistance against them. BceRS-BceAB (BceRS-AB) responds to bacitracin, plectasin, mersacidin, and actagardine. YxdJK-LM is induced by a cationic antimicrobial peptide, LL-37. The PsdRS-AB (formerly YvcPQ-RS) system responds primarily to lipid II-binding lantibiotics such as nisin and gallidermin. We characterized the psdRS-AB operon and defined the regulatory sequences within the P psdA promoter. Mutation analysis demonstrated that P psdA expression is fully PsdR dependent. The features of both the P bceA and P psdA promoters make them promising candidates as novel whole-cell biosensors that can easily be adjusted for high-throughput screening.

This work investigated the effect of N-acetyl-L-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 μM concentration. It was evidenced by a statistically significant increase of both GSH(t) and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statistically significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH(t) level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure.

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals. The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 micromol min(-1)mg(-1)) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54nrb and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1–topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the β-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.

The concept of bioisosteric replacement matrices is applied to explore the chemical space of serotonin receptor ligands, aiming to determine the most efficient ways of manipulating the affinity for all 5-HT receptor subtypes. Analysis of a collection of over 1 million bioisosteres of compounds with measured activity towards serotonin receptors revealed that an average of 31 % of the ligands for each target are mutual bioisosteres. In addition, the collected dataset allowed the development of bioisosteric matrices—qualitative and quantitative descriptions of the biological effects of each predefined type of bioisosteric substitution, providing favored paths of modifying the compounds. The concept exemplified here for serotonin receptor ligands can likely be more broadly applied to other target classes, thus representing a useful guide for medicinal chemists designing novel ligands.

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40–50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50–100 μM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N -acetyl- l -cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by l -buthionine-(S,R)-sulfoximine (BSO).

DNA relaxation catalysed by topoisomerase I is based on the reversible DNA cleavage. The reaction is inhibited by binding of splicing protein SF2/ASF, a substrate for the kinase activity of topoisomerase I. In this paper, we show a novel binding site for SF2/ASF in the cap region of topoisomerase I (amino acids 215–433) which interacts with the region containing two closely spaced RRM domains of SF2/ASF (amino acids 1–194). The sites were defined by a set of pull-down experiments with isolated recombinant polypeptides. We also indicate that the novel site is responsible for the inhibition of DNA cleavage. The polypeptide containing tandem RRM domains inhibited DNA cleavage by topoisomerase I similarly as the complete SF2/ASF. Moreover, interaction between the tandem RRM domains and the cap region was not possible in the presence of DNA.

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.

The enzymatic studies were performed to reveal a mode of activation of human topoisomerase I by a direct interaction with protein kinase CK2. In the absence of ATP CK2 kinase activated DNA relaxation about twofold. CK2alpha subunit was identified as solely responsible for the stimulation of relaxing activity by CK2 kinase. CK2 activated the relaxation only at the excess of the substrate over topoisomerase I. At the equimolar ratio of the substrate DNA and topoisomerase I the activation was not observed. There was also no effect of CK2 on camptothecin-induced cleavage of DNA by htopo I. These results identify an accelerated movement of topoisomerase I between substrate molecules as a cause of the activation of DNA relaxation by CK2 kinase.

A splicing factor SF2/ASF is a natural substrate for the kinase activity of human topoisomerase I. This study demonstrates that SF2/ASF inhibits DNA cleavage by human topoisomerase I induced by the anti-cancer agent camptothecin. The inhibition is independent of the phosphorylation status of SF2/ASF. We show that the inhibition did not result from binding of SF2/ASF to DNA that would hinder interactions between topoisomerase I and DNA. Neither it was a consequence of a loss of sensitivity of the enzyme to camptothecin. We provide evidence pointing to reduced formation of the cleavable complex in the presence of SF2/ASF as a primary reason for the inhibition. This effect of SF2/ASF is reflected by inhibition of DNA relaxation catalysed by topoisomerase I.

a b s t r a c t Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human dis- eases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway. One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H2O2 and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N 6 -ethenoadenine (A) and 3,N 4 -ethenocytosine (C), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re- ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H2O2 or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising A and C from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.

BackgroundBacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.Methods and resultsPhylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.ConclusionsOur results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.

The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1). This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb) from the circulation, as well as the induction of haem oxygenase 1 (HO1) in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism.

Eukaryotic topoisomerase I polypeptides can be partitioned into four structural domains. The function of the N-terminal domain, which is a target for serine-specific phosphorylation, has not been fully defined. The number of serine residues in the N-terminal domain of topoisomerase I from different species is inversely proportional to the number of charged amino acids in this region of the protein. The significance of this correlation is discussed in terms of a possible role for serine-specific phosphorylation in the activity of the enzyme.

Two-component systems (TCS) are major signal transduction pathways that allow bacteria to detect and respond to environmental and intracellular changes. A group of TCS has been shown to be involved in the response against antimicrobial peptides (AMPs). These TCS are characterized by the possession of intramembrane-sensing histidine kinases, and they are usually associated with ABC transporters of the peptide-7 exporter family (Pep7E). Lactobacillus casei BL23 encodes two TCS belonging to this group (TCS09 and TCS12) that are located next to two ABC transporters (ABC09 and ABC12), as well as a third Pep7E ABC transporter not genetically associated with any TCS (orphan ABC). This study addressed the involvement of modules TCS09/ABC09 and TCS12/ABC12 in AMP resistance. Results showed that both systems contribute to L. casei resistance to AMPs, and that each TCS constitutes a functional unit with its corresponding ABC transporter. Analysis of transcriptional levels showed that module 09 is required for the induction of ABC09 expression in response to nisin. In contrast, module 12 controls a wider regulon that encompasses the orphan ABC, the dlt operon ( d -alanylation of teichoid acids), and the mprF gene ( l -lysinylation of phospholipids), thereby controlling properties of the cell envelope. Furthermore, the characterization of a dltA mutant showed that Dlt plays a major role in AMP resistance in L. casei . This is the first report on the regulation of the response of L. casei to AMPs, giving insight into its ability to adapt to the challenging environments that it encounters as a probiotic microorganism.

Oxidative stress is one of the major factors leading to Maneb- and Zineb-induced disorders. The aim of this in vitro study was to examine (i) the potency of Maneb and Zineb to induce changes in antioxidant enzyme activities in Chinese hamster fibroblasts V79 cells and (ii) the role of N-acetyl-L-cysteine (NAC) in the preventing their action. Maneb increased mitochondrial superoxide dismutase (SOD2) activity but failed to affect the activity of cytoplasmic superoxide dismutase (SOD1), whereas Zineb did not change the activity of any of superoxide dismutases. The activity of catalase (CAT) was reduced only by Zineb. The activity of both glutathione peroxidases (non-Se-GPx, Se-GPx) and glutathione reductase (GR) was decreased after exposure to these agents. After NAC pre-treatment Maneb increased the activity of GR, whereas the activity of non-Se-GPx was decreased as compared to that in NAC-treated cells. On the other hand, the activity of both SODs and CAT was decreased. Zineb decreased the activity of both GPxs and SOD2 with a concomitant increase in CAT activity comparing to NAC-treated cells. The results obtained thus suggest that Zineb acts by another mechanism, than Maneb does, and that one of the mechanisms of NAC protection against Maneb or Zineb-induced effects in V79 cells is its impact on enzymatic defense. Activity of GR, SOD2, and GPxs are the most affected enzymes.

Two sublines of LY murine lymphoma, differing in sensitivity to CPT, served as source of topoisomerase I in order to compare the enzyme's properties. The activity of topoisomerase I isolated from LY-S cells of reduced sensitivity to CPT increased about 2-times more upon phosphorylation with casein kinase but was inhibited to a lesser extent upon dephosphorylation with alkaline phosphatase than the enzyme from the CPT-sensitive LY-R cells. The in vitro phosphorylation of LY-S enzyme restored its sensitivity to CPT. The in vitro incorporation of 32P into topoisomerase protein was about 1.7-times higher in LY-S than in LY-R enzyme. A reversed incorporation ratio was observed upon metabolic labelling. The level of topoisomerase I protein, determined by Western blot analysis using scleroderma anti-topoisomerase I antibodies, was about 1.5-times higher in LY-S than in LY-R cells. The level of topoisomerase I mRNA was similar in both sublines. These results indicate that the reduced sensitivity of LY-S cells to CPT is based on the lowered phosphorylation of topoisomerase I protein but does not depend on the expression of topoisomerase I gene.

Human DNA topoisomerase I (topo I) catalyzes DNA relaxation and phosphorylates SRSF1. Whereas the structure of topo I complexed with DNA has been resolved, the structure of topo I in the complex with SRSF1 and structural determinants of topo I activities in this complex are not known. The main obstacle to resolving the structure is a contribution of unfolded domains of topo I and SRSF1 in formation of the complex. To overcome this difficulty, we employed a three-step strategy: identifying the interaction regions, modeling the complex, and validating the model with biochemical methods. The binding sites in both topo I and SRSF1 are localized in the structured regions as well as in the unfolded domains. One observes cooperation between the binding sites in topo I but not in SRSF1. Our results indicate two features of the unfolded RS domain of SRSF1 containing phosphorylated residues that are critical for the kinase activity of topo I: its spatial arrangement relative to topo I and the organization of its sequence. The efficiency of phosphorylation of SRSF1 depends on the length and flexibility of the spacer between the two RRM domains that uniquely determine an arrangement of the RS domain relative to topo I. The spacer also influences inhibition of DNA nicking, a prerequisite for DNA relaxation. To be phosphorylated, the RS domain has to include a short sequence recognized by topo I. A lack of this sequence in the mutants of SRSF1 or its spatial inaccessibility in SRSF9 makes them inadequate as topo I/kinase substrates.

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis . Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.

Sensitivity to topoisomerase II inhibitors was tested at the cellular and enzyme level for two strains of mouse L5178Y lymphoma cells: resistant (LY-R) and sensitive (LY-S) to X-radiation. Differences in the susceptibility to inhibitors between LY-R and LY-S cells depended on the inhibitor used and were observed for adriamycin and VP-16, but not for mitoxantrone. On the other hand, isolated enzymes displayed the same sensitivity to all inhibitors tested regardless of the cell line. These results exclude the presence of altered topoisomerase II in LY-S cells as a possible reason for the collateral sensitivity of LY-S cells to X-radiation and topoisomerase II inhibitors.

Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to β-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS--thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 μM).

1. 1. Activity of topoisomerase I and incorporation of [ 3 H]uridine and [ 14 C]thymidine were monitored during light-induced sporulation of the slime mold Physarum polycephalun . 2. 2. A 4-fold transient increase of topoisomerase I activity but not of [ 3 H]uridine or [ 14 C]thymidine incorporation was observed after 42 hr of illumination with 6 hr impulses. 3. 3. The activity of topoisomerase I did not increase in the absence of light impulses. However, ca 5-fold increase of the activity was observed in dark when 100 μ M dibutyryl-cAMP was administered 12 hr before harvesting of plasmodia. 4. 4. Fluorodeoxyuridine and cycloheximide administered 36 hr after starting of the illumination cancelled the increase of the activity of topoisomerase I. 5. 5. After 7 days of the illumination, when fruiting bodies appeared, the activity of topoisomerase I dropped to about 15% of the initial value.

1. 1. A regulatory coupling between the rate of cellular transcription and the activity of topoisomerase I was investigated in plasmodia of Physarum polycephalum treated with fluorodeoxyuridine or nalidixic acid. 2. 2. Fluorodeoxyuridine at concentrations above 40 μg/ml lowered both the incorporation of [ 3 H]uridine and the activity of topoisomerase I to 10% of corresponding control values. 3. 3. Nalidixic acid, in the range of concentrations between 20–50 μg/ml did not inhibit the incorporation of [ 3 H]uridine but lowered the activity of topoisomerase I by about half. 4. 4. It is suggested that a coupling between the level of transcription and the activity of topoisomerase I in Physarum plasmodia involves only about a half of the topoisomerase I activity and is limited to transcription occurring on ribosomal genes.

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).

Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression.

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.

The N-terminal domain is a fragment that binds proteins and anchors topoisomerase I in the nucleolus. As a separate polypeptide, it translocates from the nucleolus to nucleoplasm upon camptothecin treatment. In this paper, we show that the translocation depends on the short fragment of the domain (residues from 1 to 67). We also present a list of proteins that specifically bind to the fragment responsible for translocation.

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH(total)/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 microM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100-150 microM). GSH(total)/GSSG ratio in the 100 microM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 microM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH(total)/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, L-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH(total)/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.

Background and aim Peroxynitrite (ONOO − ), a highly reactive species, modulates platelet activation and function. Modifications of platelet proteins induced by ONOO − may be an important factor in the pathogenesis of platelet-related diseases. The defence mechanisms against ONOO − are therefore crucial for normal cellular function. Recently, there has been an increased interest in the screening of natural products present in the diet and herbals for possible antioxidative agents (ONOO − scavengers). d-glucaro-1,4-lactone (1,4-GL), formed from d-glucaric acid (GA), a natural compound found in fruits and vegetables, possesses detoxifying and anticancerogenic properties. However, the effect of this compound on platelet activation is unknown. Methods and results We investigated the effects of 1,4-GL on nitrative and oxidative alteration of platelet proteins and lipid peroxidation caused by two strong oxidants: ONOO − and hydroperoxide (H 2 O 2 ). The action of 1,4-GL on platelet aggregation induced by adenosine diphosphate (ADP) was also studied. Exposure of platelets to ONOO − or H 2 O 2 resulted in an increase in the level of carbonyl groups (approximately three-fold and two-fold, respectively). In the presence of 1,4-GL, a significant decrease (about 50% for the highest concentration of 1,4-GL) in carbonyl group formation was observed; however the level of nitrotyrosine residues in platelets treated with ONOO − remained unchanged. Conclusions We demonstrated an inhibitory effect of 1,4-GL on lipid peroxidation in platelets treated with ONOO − (0.1mM) or H 2 O 2 (2mM). 1,4-GL inhibited platelet lipid peroxidation by about 40%. In the presence of 1,4-GL, peroxidation of plasma lipids was also reduced by about 40%. These results demonstrate that 1,4-GL possesses antioxidative properties and reduces the activation of blood platelets.