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The growth of Lactobacillus plantarum , a member of the Lactobacillus genus, which plays a crucial role in the bacterial microbiome of the gut, is significantly influenced by manganese ions. They can be safely delivered to the intestines by exploiting the chelating abilities of lactoferrin. The aim of this work was to encapsulate lactoferrin saturated with manganese ions (MnLf) in a system based on the Eudragit ® RS polymer to protect protein from degradation and manganese release in the gastric environment. The entrapment efficiency was satisfactory, reaching about 95%, and most importantly, manganese ions were not released during microparticles (MPs) formation. The release profile of the protein from the freshly prepared MPs was sustained, with less than 15% of the protein released within the first hour. To achieve similar protein release efficiency, freeze-drying was carried out in the presence of 10% ( w / v ) mannitol as a cryoprotectant for MPs frozen at -20 °C. MPs with encapsulated MnLf exhibited prebiotic activity towards Lactobacillus plantarum . More importantly, the presence of equivalent levels of manganese ions in free form in the medium, as well as chelating by lactoferrin encapsulated in MPs, had a similar impact on stimulating bacterial growth. This indicates that the bioavailability of manganese ions in our prepared system is very good.

This study presents a comprehensive structural analysis of the adducts formed upon the reaction of two Ru(III) complexes [HIsq][ trans -Ru III Cl 4 (dmso)(Isq)] ( 1 ) and [H 2 Ind][ trans -Ru III Cl 4 (dmso)(HInd)] ( 2 ) (where HInd-indazole, Isq-isoquinoline, analogs of NAMI-A) and two Ru(II) complexes, cis -[RuCl 2 (dmso) 4 ] ( c ) and trans -[RuCl 2 (dmso) 4 ] ( t ), with hen-egg white lysozyme (HEWL). Additionally, the crystal structure of an adduct of human lysozyme (HL) with ruthenium complex, [H 2 Ind][ trans -RuCl 4 (dmso)(HInd)] was solved. X-ray crystallographic data analysis revealed that all studied Ru complexes, regardless of coordination surroundings and metal center charge, coordinate to the same amino acids (His15, Arg14, and Asp101) of HEWL, losing most of their original ligands. In the case of the 2 -HL adduct, two distinct metalation sites: (i) Arg107, Arg113 and (ii) Gln127, Gln129, were identified. Crystallographic data were supported by studies of the interaction of 1 and 2 with HEWL in an aqueous solution. Hydrolytic stability studies revealed that both complexes 1 and 2 liberate the N-heterocyclic ligand under crystallization-like conditions (pH 4.5) as well as under physiological pH conditions, and this process is not significantly affected by the presence of HEWL. A comparative examination of nine crystal structures of Ru complexes with lysozyme, obtained through soaking and co-crystallization experiments, together with in-solution studies of the interaction between 1 and 2 with HEWL, indicates that the hydrolytic release of the N-heterocyclic ligand is one of the critical factors in the interaction between Ru complexes and lysozyme. This understanding is crucial in shedding light on the tendency of Ru complexes to target diverse metalation sites during the formation and in the final forms of the adducts with proteins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oszajca, Flejszar, Szura, Dróżdż, Brindell and Kurpiewska.)

Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit ® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90-95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia , showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved.

The effect of polypyridyl Ru(II) complexes on the ability of cancer cells to migrate and invade, two features important in the formation of metastases, is evaluated. In vitro studies are carried out on breast cancer cell lines, MDA-MB-231 and MCF-7, as well as melanoma cell lines A2058 and A375. Three Ru(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and as a third ligand 2,2'-bipyridine (bpy), or its derivative with either 4-[3-(2-nitro-1H-imidazol-1-yl)propyl] (bpy-NitroIm), or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moiety attached are examined. The low sub-toxic doses of the studied compounds greatly affected the cancer cells by inhibiting cell detachment, migration, invasion, transmigration, and re-adhesion, as well as increasing cell elasticity. The molecular studies revealed that the Ru(II) polypyridyl complexes impact the activity of the selected integrins and upregulate the expression of focal adhesion components such as vinculin and paxillin, leading to an increased number of focal adhesion contacts.

The use of polypyridyl Ru complexes to inhibit metastasis is a novel approach, and recent studies have shown promising results. We have reported recently that Ru (II) complexes gathering two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and the one being 2,2'-bipyridine (bpy) or its derivative with a 4-[3-(2-nitro-1H-imidazol-1-yl)propyl (bpy-NitroIm) or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moieties can alter the metastatic cascade, among others, by modulating cell adhesion properties. In this work, we show further studies of this group of complexes by evaluating their effect on HMEC-1 endothelial cells. While all the tested complexes significantly inhibited the endothelial cell migration, Ru-bpy additionally interrupted the pseudovessels formation. Functional changes in endothelial cells might arise from the impact of the studied compounds on cell elasticity and expression of proteins (vinculin and paxillin) involved in focal adhesions. Furthermore, molecular studies showed that complexes modulate the expression of cell adhesion molecules, which has been suggested to be one of the factors that mediate the activation of angiogenesis. Based on the performed studies, we can conclude that the investigated polypyridyl Ru (II) complexes can deregulate the functionality of endothelial cells which may lead to the inhibition of angiogenesis.

Hypoxia is one of the hallmarks of the tumor microenvironment and can be used in the design of targeted therapies. Cellular adaptation to hypoxic stress is regulated by hypoxia-inducible factor 1 (HIF-1). Hypoxia is responsible for the modification of cellular metabolism that can result in the development of more aggressive tumor phenotypes. Reduced oxygen concentration in hypoxic tumor cells leads to an increase in oxidoreductase activity that, in turn, leads to the activation of hypoxia-activated prodrugs (HAPs). The same conditions can convert a non-fluorescent compound into a fluorescent one (fluorescent turn off-on probes), and such probes can be designed to specifically image hypoxic cancer cells. This review focuses on the current knowledge about the expression and activity of oxidoreductases, which are relevant in the activation of HAPs and fluorescent imaging probes. The current clinical status of HAPs, their limitations, and ways to improve their efficacy are briefly discussed. The fluorescence probes triggered by reduction with specific oxidoreductase are briefly presented, with particular emphasis placed on those for which the correlation between the signal and enzyme expression determined with biochemical methods is achievable.

The purpose of this study was to investigate the correlation between the spectroscopic and photophysical properties of Ru(II) polypyridyl complexes and their photodynamic activity in vitro . A series of Ru(II) polypyridyl complexes with 4,7-diphenyl-1,10-phenanthroline (dip) and 2,3-bis(2-pyridyl)quinoxaline (dpq) and its derivatives were synthesized and characterized regarding their photophysical, biological, and photodynamic properties. The complexes were evaluated not only in the context of 1 O 2 generation but also regarding other types of reactive oxygen species (ROS) to assess the possibility of Ru(II) complexes to induce phototoxicity via various ROS using fluorescence and EPR spectroscopy. The compounds were found to be moderately cytotoxic with IC 50 values ranging from 1 to 35 μM and retained their cytotoxic activity under hypoxic conditions. The unraveled phototoxic activity is based mainly on the generation of H 2 O 2 and 1 O 2 , highlighting the importance of electron-transfer processes in the observed photodynamic activity of Ru polypyridyl complexes. A combination of photodynamic activity with cytotoxicity under decreased dioxygen concentrations may help overcome the current photodynamic therapy (PDT) limitation. The findings highlight the need for broadening the scope of tested Ru-based photosensitizers.

In recent years, Ru polypyridyl complexes have been intensively studied for their anticancer activity. The vast majority of research focuses on assessing their cytotoxic activity, as well as targeting cancer cells with them. Since the formation of metastases poses a greater risk than primary tumors, scientists recently began evaluating these compounds as potential metastasis inhibitors. This review highlights the latest achievements in this field with particular attention to the identification of the target proteins responsible for such activity. Cell migration, invasion, and adhesion are key components of metastasis, therefore understanding how they are affected by Ru polypyridyl complexes is of great importance. KEYWORDS: Ruthenium polypyridyl complexes Antimetastatic Migration Invasion Adhesion Metalloproteinases., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

For many years, natural products have played a crucial role in drug discovery and drug design as a source of active agents or as inspiration. Lactoferrin (Lf), a glycoprotein found in milk and mammalian secretions, has been extensively studied in recent years, and numerous antimicrobial, anti-inflammatory, and anticancer properties of Lf have been demonstrated in the literature. The use of lactoferrin as a co-agent or supplement to enhance the beneficial effect of drugs, or to reduce their side effects, arouses the interest of many researchers, especially since Lf is a well-studied, biocompatible, cheap, and easily accessible protein. In this mini-review, we focus on the elucidation of the role of Lf in antimicrobial or anticancer therapies, pointing to the possible mechanism underlying the determined synergism between Lf and commonly used drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Primary tumor targeting is the dominant approach in drug development, while metastasis is the leading cause of cancer death. Therefore, in addition to the cytotoxic activity of a series of Ru(II) polypyridyl complexes of the type [Ru(dip) 2 L] 2+ (dip: 4,7-diphenyl-1,10-phenanthroline while L = dip; bpy: 2,2'-bipyridine; bpy-SC: bipyridine derivative bearing a semicarbazone 2-formylopyridine moiety; dpq, dpq(CH 3 ) 2 , dpb: quinoxaline derivatives) their ability to inhibit cell detachment was investigated. In vitro studies performed on lung cancer A549 cells showed that they accumulate in cells very well and exhibit moderate cytotoxicity with IC 50 ranging from 4 to 13 µM. Three of the studied compounds that have dip, bpy-SC, or dpb ligands after treatment of the cells with a non-toxic dose (< 1 / 2 IC 50 ) enhanced their adhesion properties demonstrated by lower detachment in the trypsin resistance assay. The same complexes inhibited both MMP-2 and MMP-9 enzyme activities with IC 50 ranging from 2 to 12 µM; however, the MMP-9 inhibition was stronger. More detailed studies for [Ru(dip) 2 (bpy-SC)] 2+ , which induced the greatest increase in cell adhesion, revealed that it is predominately accumulated in the cytoskeletal fraction of A549 cells. Moreover, cells treated with this compound showed the localization of MMP-9 to a greater extent also in the cytoskeleton. Taken together, our results indicate the possibility of a reduction of metastatic cells escaping from the primary lesion to the surrounding tissue by prevention of their detachment and by influencing the activity of MMP-2 and MMP-9.

One of the consequences of long-term exposure to air pollutants is increased mortality and deterioration of life parameters, especially among people diagnosed with cardiovascular diseases (CVD) or impaired respiratory system. Aqueous soluble inorganic components of airborne particulate matter containing redox-active transition metal ions affect the stability of S-nitrosothiols and disrupt the balance in the homeostasis of nitric oxide. Blood plasma's protective ability against the decomposition of S-nitrosoglutathione (GSNO) under the influence of aqueous PM extract among patients with exacerbation of heart failure and coronary artery disease was studied and compared with a group of healthy volunteers. In the environment of CVD patients' plasma, NO release from GSNO was facilitated compared to the plasma of healthy controls, and the addition of ascorbic acid boosted this process. Model studies with albumin revealed that the amount of free thiol groups is one of the crucial factors in GSNO decomposition. The correlation between the concentration of NO released and -SH level in blood plasma supports this conclusion. Complementary studies on gamma-glutamyltranspeptidase activity and ICP-MS multielement analysis of CVD patients' plasma samples in comparison to a healthy control group provide broader insights into the mechanism of cardiovascular risk development induced by air pollution.

S-Nitrosothiols act as a comparatively long-lived reservoir of releasable nitric oxide (NO) present in vivo in a variety of body fluids. Soluble constituents of air-borne particulate matter (PM) can affect S-nitrosothiol stability and deregulate NO-based biological signaling. PM aqueous extracts of standard urban dust (SRM 1648a) were prepared, and their effect on human serum S-nitrosoalbumin (HSA-NO) stability was studied. The results indicated that PM extracts induced a release of NO from HSA-NO in a dose-dependent manner. To identify the inorganic components of urban PM responsible for HSA-NO decomposition, the effects of individual metal ions and metal ion mixtures, detected in the SRM 1648a aqueous extract, were examined. The dominant role of copper ions (specifically Cu+) was confirmed, but the results did not exclude the influence of other water-soluble PM components. Measurements with the application of several common metal ion chelators confirmed that Cu2+ may participate in NO release from HSA-NO and that reduction to monovalent Cu+ (responsible for S-NO bond breaking) may occur with the participation of S-nitrosoalbumin. The addition of ascorbic acid (AscA) significantly enhanced the effectiveness of NO release by PM extracts both kinetically and quantitatively, by inducing an increase in the reduction of Cu2+ to Cu+. These results indicate that AscA present in the respiratory tract lining fluids and plasma may amplify the activity of inorganic components of PM in S-nitrosothiol decomposition.

Air pollution is associated with numerous negative effects on human health. The toxicity of organic components of air pollution is well-recognized, while the impact of their inorganic counterparts in the overall toxicity is still a matter of various discussions. The influence of airborne particulate matter (PM) and their inorganic components on biological function of human alveolar-like epithelial cells (A549) was investigated in vitro . A novel treatment protocol based on covering culture plates with PM allowed increasing the studied pollutant concentrations and prolonging their incubation time without cell exposure on physical suffocation and mechanical disturbance. PM decreased the viability of A549 cells and disrupted their mitochondrial membrane potential and calcium homeostasis. For the first time, the difference in the reactive oxygen species (ROS) profiles generated by organic and inorganic counterparts of PM was shown. Singlet oxygen generation was observed only after treatment of cells with inorganic fraction of PM, while hydrogen peroxide, hydroxyl radical, and superoxide anion radical were induced after exposure of A549 cells to both PM and their inorganic fraction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Mazuryk, Stochel and Brindell.)

The well-documented cytotoxic activity of coordinatively saturated and substitutionally inert polypyridyl Ru(ii) complexes substantiates their high potency as antiproliferative agents against primary tumors. However, the primary cause of cancer morbidity and mortality responsible for about 90% of cancer deaths is the occurrence of metastasis. Therefore, scientists have to concentrate their efforts on designing compounds affecting not only the primary tumor, but also efficiently inhibiting metastasis. Herein, we report two families of Ru(ii) polypyridyl complexes bearing 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety as one of the ligands and 4,4'-di-tert-butyl-2,2'-dipyridyl or 4,7-diphenyl-1,10-phenanthroline as auxiliary ligands. These complexes strengthen cells' adherent properties and inhibit the activity of metalloproteinases (MMPs) in vitro, which is relevant in anti-metastatic treatment. The in vitro studies were performed on human lung adenocarcinoma (A549) and human pancreatic cancer (PANC-1) cells, which have a well-documented invasive potential. The induced alteration of the tumor cells' adhesion properties correlated with the high cytotoxic effect exerted by the complexes and their excellent cellular uptake. It was also proved that both complexes directly inhibit M-MP2 and M-MP9 enzyme activities, which are essential for the development of tumor metastasis. The results of this study indicate that the biological properties of polypyridyl Ru(ii) complexes extend beyond the standard cytotoxic activity and represent an important step towards designing new anti-metastatic agents.

Exposure to airborne particulate matter (PM) is associated with hazardous effects on human health. Soluble constituents of PM may be released in biological fluids and disturb the precisely tuned nitric oxide signaling processes. The influence of aqueous extracts from two types of airborne urban PM (SRM 1648a, a commercially available sample, and KR PM2.5, a sample collected "in-house" in Krakow, Poland) on the stability of S-nitrosoglutathione (GSNO) was investigated. The particle interfaces had no direct effect on the studied reaction, but extracts obtained from both samples facilitated NO release from GSNO. The effectiveness of NO release was significantly affected by glutathione (GSH) and ascorbic acid (AscA). Examination of the combined influence of Cu 2+ , Fe 3+ , and reductants on GSNO stability revealed copper to be the main GSNO decomposing species. Computational models of nitrosothiols interacting with metal oxide substrates and solvated metal ions support these claims. The study stresses the importance of the interplay between metal ions and biological reductants in S-nitrosothiols decomposition., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lactoferrin was isolated and purified for the first time over 50-years ago. Since then, extensive studies on the structure and function of this protein have been performed and the research is still being continued. In this mini-review we focus on presenting recent scientific efforts towards the elucidation of the role and therapeutic potential of lactoferrin saturated with iron(III) or manganese(III) ions. The difference in biological activity of metal-saturated lactoferrin vs. the unmetalated one is emphasized. The strategies for oral delivery of lactoferrin, are also reviewed, with particular attention to the metalated protein., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ruthenium(II) polypyridyl complexes have been extensively studied for the past few decades as promising anticancer agents. Despite the expected intravenous route of administration, the interaction between Ru(II) polypyridyl compounds and serum proteins is not well characterized and vast majority of the available literature data concerns determination of the binding constant. Ru-protein adducts can modify the biological effects of the Ru complexes influencing their cytotoxic and antimicrobial activity as well as introduce significant changes in their photophysical properties. More extensive research on the interaction between serum proteins and Ru(II) polypyridyl complexes is important for further development of Ru(II) polypyridyl compounds towards their application in anticancer therapy and diagnostics and can open new opportunities for already developed complexes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Two ruthenium(II) complexes Ru1 and Ru2 bearing as a one ligand 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety (bpySC: 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone) and as the others 2,2'-bipyridine (bpy) and 4,7-diphenyl-1,10-phenanthroline (dip), respectively, as auxiliary ligands have been prepared. Their biological activity has been studied on murine colon carcinoma (CT26) and human lung adenocarcinoma (A549) cell lines. The anti-proliferative activity was dependent on the presence of bpy or dip in the complex, with one order of magnitude higher cytotoxicity for Ru2 (dip ligands). Ru1 (bpy ligands) exhibited a distinct increase in cytotoxicity going from 24 to 72h of incubation with cells as was not observed for Ru2. Even though both studied compounds were powerful apoptosis inducing agents, the mechanism of their action was entirely different. Ru1-incubated A549 cells showed a notable increase in cells number in the S-phase of the cell cycle, with concomitant decrease in the G2/M phase, while Ru2 promoted a cell accumulation in the G0/G1 phase. In contrast, Ru1 induced marginal oxidative stress in A549 cell lines even upon increasing the incubation time. Even though Ru1 preferably accumulated in lysosomes it triggered the apoptotic cellular death via an intrinsic mitochondrial pathway. Ru1-incubated A549 cells showed swelling and enlarging of the mitochondria. It was not observed in case of Ru2 for which mitochondria and endoplasmic reticulum were found as primarily localization site. Despite this the apoptosis induced by Ru2 was caspase-independent. All these findings point to a pronounced role of auxiliary ligands in tuning the mode of biological activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The mechanism of cell death induced by the ruthenium polypyridyl complexes comprising two 4,7-diphenyl-1,10-phenanthroline ligands as well as one unmodified 2,2'-bipyridyl or modified with 2-nitroimidazole moiety attached by shorter (C 3 H 6 ) or longer (C 6 H 12 ) linker was investigated. Cytotoxicity and proliferation assays revealed that the studied Ru polypyridyl complexes are more toxic against human pancreas carcinoma PANC-1 cell line than normal human keratinocytes HaCaT with IC 50 of 3-5μM. The Ru complexes despite accumulation in mitochondria do not lead to mitochondrial disfunction, though decreasing of mitochondrial Ca 2+ causes mitochondria membrane hyperpolarization. The Ru polypyridyl conjugates induce some phenotypical characteristic of apoptosis, such as condensation of chromatin or phosphatidylserine translocation, however no caspase or calpain activation in the studied cell lines was observed, indicating that detected cell death does not occur via mitochondria- or ER-activated pathways. Caspase-independent cell death is caused by enormous ROS formation, mainly hydrogen peroxide and peroxyl radicals as well as by intracellular Ca 2+ homeostasis disruption. Accumulation of the Ru compounds inhibits the completion of DNA synthesis, arresting cells in S-phase of cell cycle., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The antimetastatic ruthenium(III) complex (H 2 Im)[trans-RuCl 4 (HIm)(DMSO)] (NAMI-A) as well as its two analogues (H 2 Ind)[trans-RuCl 4 (HInd)(DMSO)] (Ru-Ind) and (HIsq)[trans-RuCl 4 (Isq)(DMSO)] (Ru-Isq) (HIm-imidazole, HInd-indazole, Isq-isoquinoline, DMSO-dimethyl sulfoxide) were tested for their effect on endothelial cell functions in vitro on human skin microvascular endothelial cells (HSkMEC) and human endothelial progenitor cells (HPEC-CB.2) under normoxic (21 % O 2 ) and hypoxic (1 % O 2 ) conditions. All studied complexes showed very low cytotoxicity profiles towards both mature microvascular and precursor endothelial cells (ECs), independently of oxygen concentration. Among tested compounds Ru-Ind exhibited the highest cytotoxicity. The antiangiogenic activity of ruthenium complexes was evaluated for their influence on pseudo-vessels formation by microvascular endothelial cells (HSkMEC) because of their involvement in melanoma progression. Our studies indicated that Ru-Ind and Ru-Isq exhibited hypoxia- and dose-dependent-inhibition of angiogenesis on Matrigel™. Significant hypoxia-selective downregulation of pseudo-vessels formation by Ru-Isq correlates with efficient inhibition of cell motility. Interestingly, in the applied concentration doses migration of endothelial cells was also inhibited by NAMI-A, but the pseudo-vessels formation on Matrigel™ was unaffected. Angiogenesis-related genes expression profile for both mature and precursor ECs indicated that inhibition of angiogenesis, mainly due to Ru-Isq, as compared to NAMI-A and Ru-Ind correlated with downregulation of CD31 and CD144 expression and upregulation of NOTCH4 expression in mature ECs, which is essential for endothelial cell motility and stalk cells organization control. The hypoxia-selective antiangiogenic activity of Ru-Ind and Ru-Isq, NAMI-A analogues makes them potent antimetastatic therapeutics for their selective action in hypoxia which controls tumor pathologic angiogenesis.

Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis., Competing Interests: The authors declare that they have no conflict of interest.

The ruthenium polypyridyl complexes [Ru(dip)2(bpy/bpy-2-nitroIm)](2+) (dip = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2'-bipyridine, bpy-2-nitroIm = 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]) were found to be ca. ten times more cytotoxic against breast cancer (4T1) and human lung adenocarcinoma epithelial cells (A549) than a well-known anticancer drug, cisplatin. Even though the Ru complexes were quite cytotoxic towards FVB mouse lung microvascular endothelial cells (MLuMEC FVB) their efflux from these non transformed cells was much more efficient than from cancer ones. Both Ru complexes accumulated in cells. The cellular uptake of both Ru complexes occurs through passive diffusion while the nitroimidazole derivative is also endocytosed. They arrest cell growth in the S-phase and induce apoptosis. Such cell response can result from activation of oxidative stress by Ru complexes. The modulation of the mRNA expression profile for genes which might be involved in metastasis and angiogenesis processes by Ru complexes was analyzed for both cancer (4T1) and endothelial (MLuMEC FVB) cells. Ru complexes appeared to have a distinct impact on cell adhesion and migration as well as they affect endothelial cell vasculature. They are not only cytotoxic but are also potentially invasive and anti-metastatic agents. This work illustrates the putative future development of polypyridyl ruthenium.

Here, we examine the photophysical properties of five ruthenium(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and functionalized bipyridine (R₁bpy-R₂, where R₁= H or CH3, R₂= H, CH₃, COO⁻,4-[3-(2-nitro-1H-imidazol-1-yl)propyl] or 1,3-dicyclohexyl-1-carbonyl-urea) towards development of luminescence probes for cellular imaging. These complexes have been shown to interact with albumin and the formed adducts exhibited up to eightfold increase in the luminescence quantum yield as well as the average lifetime of emission. It was demonstrated that they cannot bind to DNA through the intercalation mode and its luminescence in the presence of DNA is quenching. Cell viability experiments indicated that all complexes possess significant dose-dependent cytotoxicity (with IC₅₀ 5-19 μM) on 4T1 breast cancer cell line and their anti-proliferative activity correlates very well with their lipophilicity. Cellular uptake was studied by measuring the ruthenium content in cells using ICP-MS technique. As expected, the better uptake is directly related to higher lipophilicity of doubly charged ruthenium complexes while uptake of monocationic one is much lower in spite of the highest lipophilicity. Additionally staining properties were assessed using flow cytometry and fluorescence microscopy. These experiments showed that complex with 1,3-dicyclohexyl-1-carbonyl-urea substituent exhibits the best staining properties in spite of the lowest luminescence quantum yield in buffered solution (pH 7.4). Our results point out that both the imaging and cytotoxic properties of the studied ruthenium complexes are strongly influence by the level of internalization and protein interaction.

For the exploration of the intrinsic reactivity of two key active species in the catalytic cycle of horseradish peroxidase (HRP), Compound I (HRP-I) and Compound II (HRP-II), we generated in situ [Fe(IV) O(TMP(+.) )(2-MeIm)](+) and [Fe(IV) O(TMP)(2-MeIm)](0) (TMP=5,10,15,20-tetramesitylporphyrin; 2-MeIm=2-methylimidazole) as biomimetics for HRP-I and HRP-II, respectively. Their catalytic activities in epoxidation, hydrogen abstraction, and heteroatom oxidation reactions were studied in acetonitrile at -15 °C by utilizing rapid-scan UV/Vis spectroscopy. Comparison of the second-order rate constants measured for the direct reactions of the HRP-I and HRP-II mimics with the selected substrates clearly confirmed the outstanding oxidizing capability of the HRP-I mimic, which is significantly higher than that of HRP-II. The experimental study was supported by computational modeling (DFT calculations) of the oxidation mechanism of the selected substrates with the involvement of quartet and doublet HRP-I mimics ((2,4) Cpd I) and the closed-shell triplet spin HRP-II model ((3) Cpd II) as oxidizing species. The significantly lower activation barriers calculated for the oxidation systems involving (2,4) Cpd I than those found for (3) Cpd II are in line with the much higher oxidizing efficiency of the HRP-I mimic proven in the experimental part of the study. In addition, the DFT calculations show that all three reaction types catalyzed by HRP-I occur on the doublet spin surface in an effectively concerted manner, whereas these reactions may proceed in a stepwise mechanism with the HRP-II mimic as oxidant. However, the high desaturation or oxygen rebound barriers during CH bond activation processes by the HRP-II mimic predict a sufficient lifetime for the substrate radical formed through hydrogen abstraction. Thus, the theoretical calculations suggest that the dissociation of the substrate radical may be a more favorable pathway than desaturation or oxygen rebound processes. Importantly, depending on the electronic nature of the oxidizing species, that is, (2,4) Cpd I or (3) Cpd II, an interesting region-selective conversion phenomenon between sulfoxidation and H-atom abstraction was revealed in the course of the oxidation reaction of dimethylsulfide. The combined experimental and theoretical study on the elucidation of the intrinsic reactivity patterns of the HRP-I and HRP-II mimics provides a valuable tool for evaluating the particular role of the HRP active species in biological systems., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed. Using a surface response methodology and a three-factorial Doehlert design we have established a new optimized running buffer composition: 50mM Tris-HCl, pH 8.5, 22.5% (v/v) methanol, 17.5mM SDS. As a result, two previously unobserved monoferric forms of protein have been separated and identified, moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerably reduced by methanol, and the method selectivity has been yet increased resulting in a total separation of proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowed us to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein with the buffers with different concentrations of ferric ions. It revealed continuously changing contribution of monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobes of protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside the capillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion mediated and electrophoretically mediated ones. Finally, the total time of separation has been decreased down to 4min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel long-lifetime highly luminescent ruthenium polypyridyl complex containing 2-nitroimidazole moiety [Ru(dip)2(bpy-2-nitroIm)]Cl2 (dip=4,7-diphenyl-1,10-phenanthroline, bpy-2-nitroIm=4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine) has been designed cancer treatment and imaging. The luminescence properties of the synthesized compound strongly depend on the oxygen concentration. Under oxygen-free conditions quantum yield of luminescence and the average lifetime of emission were found to be 0.034 and 1.9 μs, respectively, which is ca. three times higher in comparison to values obtained in air-equilibrated solution. The binding properties of the investigated ruthenium complex to human serum albumin have been studied and the apparent binding constant for the formation of the protein-ruthenium adduct was determined to be 1.1×10(5)M(-1). The quantum yield and the average lifetime of emission are greatly enhanced upon binding of ruthenium compound to the protein. The DNA binding studies revealed two distinguished binding modes which lead to a decrease in luminescence intensity of ruthenium complex up to 60% for [DNA]/[Ru]<2, and enhancement of emission for [DNA]/[Ru]>80. Preliminary biological studies confirmed fast and efficient accumulation of the ruthenium complex inside cells. Furthermore, the ruthenium complex was found to be relatively cytotoxic with LD50 of 12 and 13 μM for A549 and CT26 cell lines, respectively, under normoxic conditions. The retention and cellular uptake of ruthenium complex is enhanced under hypoxic conditions and its LD50 decreases to 8 μM for A549 cell line., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The presented results cover a comparative mechanistic study on the reactivity of compound (Cpd) I and II mimics of a water-soluble iron(III) porphyrin, [meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III), Fe(III)(TMPS). The acidity of the aqueous medium strongly controls the chemical nature and stability of the high-valent iron(IV) oxo species. Reactivity studies were performed at pH 5 and 10, where the Cpd I and II mimics are stabilized as the sole oxidizing species, respectively. The contributions of ΔH(‡) and ΔS(‡) to the free energy of activation (ΔG(‡)) for the oxidation of 4-methoxybenzaldehyde (4-MB-ald), 4-methoxybenzyl alcohol (4-MB-alc), and 1-phenylethanol (1-PhEtOH) by the Cpd I and II mimics were determined. The relatively large contribution of the ΔH(‡) term in comparison to the -TΔS(‡) term to ΔG(‡) for reactions involving the Cpd II mimic indicates that the oxidation of selected substrates by this oxidizing species is clearly an enthalpy-controlled process. In contrast, different results were found for reactions with application of the Cpd I mimic. Depending on the nature of the substrate, the reaction at room temperature can be entropy-controlled, as found for the oxidation of 4-MB-alc, or enthalpy-controlled, as found for 1-PhEtOH. Importantly, for the first time, activation volumes (ΔV(‡)) for the oxidation of selected substrates by both reactive intermediates could be determined. Positive values of ΔV(‡) were found for reactions with the Cpd II mimic and slightly negative ones for reactions with the Cpd II mimic. The results are discussed in the context of the oxidation mechanism conducted by the Cpd I and II mimics.

High-valent iron-oxo species have been invoked as reactive intermediates in catalytic cycles of heme and nonheme enzymes. The studies presented herein are devoted to the formation of compound II model complexes, with the application of a water soluble (TMPS)Fe(III)(OH) porphyrin ([meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III) hydroxide) and hydrogen peroxide as oxidant, and their reactivity toward selected organic substrates. The kinetics of the reaction of H2O2 with (TMPS)Fe(III)(OH) was studied as a function of temperature and pressure. The negative values of the activation entropy and activation volume for the formation of (TMPS)Fe(IV)=O(OH) point to the overall associative nature of the process. A pH-dependence study on the formation of (TMPS)Fe(IV)=O(OH) revealed a very high reactivity of OOH(-) toward (TMPS)Fe(III)(OH) in comparison to H2O2. The influence of N-methylimidazole (N-MeIm) ligation on both the formation of iron(IV)-oxo species and their oxidising properties in the reactions with 4-methoxybenzyl alcohol or 4-methoxybenzaldehyde, was investigated in detail. Combined experimental and theoretical studies revealed that among the studied complexes, (TMPS)Fe(III)(H2O)(N-MeIm) is highly reactive toward H2O2 to form the iron(IV)-oxo species, (TMPS)Fe(IV)=O(N-MeIm). The latter species can also be formed in the reaction of (TMPS)Fe(III)(N-MeIm)2 with H2O2 or in the direct reaction of (TMPS)Fe(IV)=O(OH) with N-MeIm. Interestingly, the kinetic studies involving substrate oxidation by (TMPS)Fe(IV)=O(OH) and (TMPS)Fe(IV)=O(N-MeIm) do not display a pronounced effect of the N-MeIm axial ligand on the reactivity of the compound II mimic in comparison to the OH(-) substituted analogue. Similarly, DFT computations revealed that the presence of an axial ligand (OH(-) or N-MeIm) in the trans position to the oxo group in the iron(IV)-oxo species does not significantly affect the activation barriers calculated for C-H dehydrogenation of the selected organic substrates., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A(280/466)). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.

NAMI-A i.e. (ImH)[trans-RuCl(4)(DMSO)(Im)] (where Im is imidazole) is a ruthenium(III) complex with promising antimetastatic activity, which has been classified for II phase clinical trial. In this study, its binding properties toward apo-transferrin (apo-Tf) with regard to its hydrolytic and redox behavior are systematically investigated by the use of fluorescence spectroscopy. The reaction of NAMI-A and its reduced form with apo-Tf is proceeded by formation of aqua derivatives and the presence of at least one labile aqua ligand is sufficient to form adducts. It is found that presence of bicarbonate is not necessary for interaction of studied ruthenium complexes with apo-Tf. The calculated association constants for both NAMI-A and its reduced form are very similar with the values of 1.28 × 10(4)M(-1) and 1.36 × 10(4)M(-1) at 37 °C, respectively however, the reduced derivatives reach the equilibrium ca. 8-10 times slower. The percentage of ruthenium content in protein fractions separated from protein-unbounded ruthenium by using FPLC (fast protein liquid chromatography) method is rather high and depends on redox state of the complex, for most samples is found higher for reduced species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Urease, a Ni-containing metalloenzyme, features an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme (jack bean), K (M) and k (cat), measured at different temperatures and pressures. Such an analysis is useful as it provides information on the molecular nature of the intermediate and transition states of the catalytic reaction. We measured the parameters in a noninteracting buffer using a stopped-flow technique in the temperature range 15-35 °C and in the pressure range 5-132 MPa, the pressure-dependent measurements being the first of their kind performed for urease. While temperature enhanced the activity of urease, pressure inhibited the enzyme; the inhibition was biphasic. Analyzing K (M) provided the characteristics of the formation of the ES complex, and analyzing k (cat), the characteristics of the activation of ES. From the temperature-dependent measurements, the energetic parameters were derived, i.e. thermodynamic ΔH (o) and ΔS (o) for ES formation, and kinetic ΔH ( ≠ ) and ΔS ( ≠ ) for ES activation, while from the pressure-dependent measurements, the binding ΔV (b) and activation ΔV(#)(cat) volumes were determined. The thermodynamic and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al. (Structure 7:205-216; 1999), in which the Ni-Ni bridging hydroxide--not the terminal hydroxide--is the nucleophile in the catalytic reaction.

In this work, we studied the behaviours of urease in the presence of l-ascorbic acid (AA) and dehydroascorbic acid (DHA) in different conditions. The inactivations of urease were carried out in an unbuffered and buffered system. We show that in the unbuffered system AA inactivated urease in a biphasic manner by denaturation brought about by AA-lowered pH. Further, we show that in the buffered system neither AA nor DHA themselves are inhibitors of urease. The inhibitory action of AA and DHA was revealed in the presence of Fe(3+) ions and most importantly, unlike reported in the literature, it was found to be primarily mediated by H(2)O(2). The resulting inhibition by DHA-Fe(3+) consisted of enzyme thiol oxidation and its effectiveness grew with increasing pH. The results may shed light on the roles of AA in therapies applied in ureolytic bacteria infections, notably those with Helicobacter pylori.

The reaction of the water-soluble Fe(III)(TMPS) porphyrin with CN(-) in basic solution leads to the stepwise formation of Fe(III)(TMPS)(CN)(H(2)O) and Fe(III)(TMPS)(CN)(2). The kinetics of the reaction of CN(-) with Fe(III)(TMPS)(CN)(H(2)O) was studied as a function of temperature and pressure. The positive value of the activation volume for the formation of Fe(III)(TMPS)(CN)(2) is consistent with the operation of a dissociatively activated mechanism and confirms the six-coordinate nature of the monocyano complex. A good agreement between the rate constants at pH 8 and 9 for the formation of the dicyano complex implies the presence of water in the axial position trans to coordinated cyanide in the monocyano complex and eliminates the existence of Fe(III)(TMPS)(CN)(OH) under the selected reaction conditions. Both Fe(III)(TMPS)(CN)(H(2)O) and Fe(III)(TMPS)(CN)(2) bind nitric oxide (NO) to form the same nitrosyl complex, namely, Fe(II)(TMPS)(CN)(NO(+)). Kinetic studies indicate that nitrosylation of Fe(III)(TMPS)(CN)(2) follows a limiting dissociative mechanism that is supported by the independence of the observed rate constant on [NO] at an appropriately high excess of NO, and the positive values of both the activation parameters ΔS(‡) and ΔV(‡) found for the reaction under such conditions. The relatively small first-order rate constant for NO binding, namely, (1.54 ± 0.01) × 10(-2) s(-1), correlates with the rate constant for CN(-) release from the Fe(III)(TMPS)(CN)(2) complex, namely, (1.3 ± 0.2) × 10(-2) s(-1) at 20 °C, and supports the proposed nitrosylation mechanism.

This article focuses on the application of high pressure laser flash photolysis for studies on selected hemoprotein reactions with the objective to establish details of the underlying reaction mechanisms. In this context, particular attention is given to the reactions of small molecules such as dioxygen, carbon monoxide, and nitric oxide with selected hemoproteins (hemoglobin, myoglobin, neuroglobin and cytochrome P450(cam)), as well as to photo-induced electron transfer reactions occurring in hemoproteins (particularly in various types of cytochromes). Mechanistic conclusions based on the interpretation of the obtained activation volumes are discussed in this account.

The cytotoxicity and photocytotoxicity of trans-[RuCl2(DMSO)4] and cis-[RuCl2(DMSO)4] complexes was tested in two melanoma cell lines, human (SK-MEL 188) and mouse (S91). The trans isomer was found to be more effective for cell growth inhibition than its cis analogue both in the presence and in the absence of illumination. However, the antiproliferative activity of both isomers was significantly enhanced after irradiation with UVA light in comparison with their activity observed in the dark. The influence of light on the reaction of both ruthenium(II) isomers with the single-stranded hexanucleotide d(T2GGT2), chosen as a model system for DNA, was also studied using chromatography and mass spectrometry techniques. The photochemical reaction of the ruthenium(II) complexes with the oligonucleotide d(T2GGT2) resulted in the formation of Ru(G-N7)2 adducts, which was not observed in the same time scale in thermal reactions. The initial short irradiation of the inert cis isomer was found to facilitate the covalent adduct formation with d(T2GGT2) in the secondary thermal reactions and with a rate comparable to that found for the trans isomer, which is ca. 5-10 times more reactive in the dark.