Expanding Mn2+ loading capacity of BSA via mild non-thermal denaturing and cross-linking as a tool to maximize the relaxivity of water protons.

Development of nanoparticles (NPs) serving as contrast enhancing agents in MRI requires a combination of high contrasting effect with the biosafety and hemocompatibility. This work demonstrates that bovine serum albumin (BSA) molecules bound to paramagnetic Mn2+ ions are promising building blocks of such NPs. The desolvation-induced denaturation of BSA bound with Mn2+ ions followed by the glutaraldehyde-facilitated cross-linking provides the uniform in size 102.0 ± 0.7 nm BSA-based nanoparticles (BSA-NPs) loaded with Mn2+ ions, which are manifested in aqueous solutions as negatively charged spheres with high colloid stability. The optimal loading of Mn2+ ions into BSA-NPs provides maximum values of longitudinal and transverse relaxivity at 98.9 and 133.6 mM-1 s-1, respectively, which are among the best known from the literature. The spin trap EPR method indicates that Mn2+ ions bound to BSA-NPs exhibit poor catalytic activity in the Fenton-like reaction. On the contrary, the presence of BSA-NPs has an antioxidant effect by preventing the accumulation of hydroxyl radicals produced by H2O2. The NPs exhibit remarkably low hemolytic activity and hemagglutination can be avoided at concentrations lower than 110 µM. Thus, BSA-NPs bound with Mn2+ ions are promising candidates for combining high contrast effect with biosafety and hemocompatibility.

TimeTeller: A tool to probe the circadian clock as a multigene dynamical system.

Recent studies have established that the circadian clock influences onset, progression and therapeutic outcomes in a number of diseases including cancer and heart diseases. Therefore, there is a need for tools to measure the functional state of the molecular circadian clock and its downstream targets in patients. Moreover, the clock is a multi-dimensional stochastic oscillator and there are few tools for analysing it as a noisy multigene dynamical system. In this paper we consider the methodology behind TimeTeller, a machine learning tool that analyses the clock as a noisy multigene dynamical system and aims to estimate circadian clock function from a single transcriptome by modelling the multi-dimensional state of the clock. We demonstrate its potential for clock systems assessment by applying it to mouse, baboon and human microarray and RNA-seq data and show how to visualise and quantify the global structure of the clock, quantitatively stratify individual transcriptomic samples by clock dysfunction and globally compare clocks across individuals, conditions and tissues thus highlighting its potential relevance for advancing circadian medicine.

Ceruloplasmin Reduces the Lactoferrin/Oleic Acid Antitumor Complex-Mediated Release of Heme-Containing Proteins from Blood Cells.

Our previous study showed that not only bovine lactoferrin (LF), the protein of milk and neutrophils, but also the human species forms complexes with oleic acid (OA) that inhibit tumor growth. Repeated injections of human LF in complex with OA (LF/8OA) to hepatoma-carrying mice decelerated tumor growth and increased animals' longevity. However, whether the effect of the LF/8OA complex is directed exclusively against malignant cells was not studied. Hence, its effect on normal blood cells was assayed, along with its possible modulation of ceruloplasmin (CP), the preferred partner of LF among plasma proteins. The complex LF/8OA (6 µM) caused hemolysis, unlike LF alone or BSA/8OA (250 µM). The activation of neutrophils with exocytosis of myeloperoxidase (MPO), a potent oxidant, was induced by 1 µM LF/8OA, whereas BSA/8OA had a similar effect at a concentration increased by an order. The egress of heme-containing proteins, i.e., MPO and hemoglobin, from blood cells affected by LF/8OA was followed by a pronounced oxidative/halogenating stress. CP, which is the natural inhibitor of MPO, added at a concentration of 2 mol per 1 mol of LF/8OA abrogated its cytotoxic effect. It seems likely that CP can be used effectively in regulating the LF/8OA complex's antitumor activity.

New Application of the Commercially Available Dye Celestine Blue B as a Sensitive and Selective Fluorescent "Turn-On" Probe for Endogenous Detection of HOCl and Reactive Halogenated Species.

Hypochlorous acid (HOCl) derived from hydrogen peroxide and chloride anion by myeloperoxidase (MPO) plays a significant role in physiological and pathological processes. Herein we report a phenoxazine-based fluorescent probe Celestine Blue B (CB) that is applicable for HOCl detection in living cells and for assaying the chlorinating activity of MPO. A remarkable selectivity and sensitivity (limit of detection is 32 nM), along with a rapid "turn-on" response of CB to HOCl was demonstrated. Furthermore, the probe was able to detect endogenous HOCl and reactive halogenated species by fluorescence spectroscopy, confocal microscopy, and flow cytometry techniques. Hence, CB is a promising tool for investigating the role of HOCl in health and disease and for screening the drugs capable of regulating MPO activity.

Caught red handed: modeling and confirmation of the myeloperoxidase ceruloplasmin alpha-thrombin complex.

The work is devoted to the study of the structural characteristics of the myeloperoxidase-ceruloplasmin-thrombin complex using small-angle neutron scattering methods in combination with computer modeling, as well as surface plasmon resonance and solid-phase enzyme assay. We have previously shown that the functioning of active myeloperoxidase during inflammation, despite the presence in the blood of an excess of ceruloplasmin which inhibits its activity, is possible due to the partial proteolysis of ceruloplasmin by thrombin. In this study, the myeloperoxidase-ceruloplasmin-thrombin heterohexamer was obtained in vitro. The building of a heterohexamer full-atomic model in silico, considering the glycosylation of the constituent proteins, confirmed the absence of steric barriers for the formation of protein-protein contacts. It was shown that the partial proteolysis of ceruloplasmin does not affect its ability to bind to myeloperoxidase, and a structural model of the heterohexamer was obtained using the small-angle neutron scattering method.

Lactoferrin Induces Erythropoietin Synthesis and Rescues Cognitive Functions in the Offspring of Rats Subjected to Prenatal Hypoxia.

The protective effects of recombinant human lactoferrin rhLF (branded "CAPRABEL™") on the cognitive functions of rat offspring subjected to prenatal hypoxia (7% O2, 3 h, 14th day of gestation) have been analyzed. About 90% of rhLF in CAPRABEL was iron-free (apo-LF). Rat dams received several injections of 10 mg of CAPRABEL during either gestation (before and after the hypoxic attack) or lactation. Western blotting revealed the appearance of erythropoietin (EPO) alongside the hypoxia-inducible factors (HIFs) in organ homogenates of apo-rhLF-treated pregnant females, their embryos (but not placentas), and in suckling pups from the dams treated with apo-rhLF during lactation. Apo-rhLF injected to rat dams either during pregnancy or nurturing the pups was able to rescue cognitive deficits caused by prenatal hypoxia and improve various types of memory both in young and adult offspring when tested in the radial maze and by the Novel Object Recognition (NOR) test. The data obtained suggested that the apo-form of human LF injected to female rats during gestation or lactation protects the cognitive functions of their offspring impaired by prenatal hypoxia.

Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity.

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

Familial Hypercholesterolemia in Russia: Three Decades of Genetic Studies.

The first studies of familial hypercholesterolemia (FH) in Russia go back to late 1980-ies. For more than 10 years the research in this field was carried out in Saint-Petersburg, the megapolis in the North-West Russia. Studies were focused on the search for causative mutations in low-density lipoprotein receptor gene (LDLR). Gradually the research was spread to Petrozavodsk in Karelia and in the XXI century two more centers contributed in investigations of genetics of FH, i.e., in Moscow and Novosibirsk. The best studied is the spectrum of mutations in LDLR, though genetic abnormalities in APOB and PCSK9 genes were also considered. Despite that some 40% mutations in LDLR found in Saint-Petersburg and Moscow are referred to as specific for Russian population, and this proportion is even higher in Karelia (ca. 70%), rapid introduction of NGS and intensifying genetic research all over the world result in continuous decrease of these numbers as "Slavic" mutations become documented in other countries. The samplings of genetically characterized patients in Russia were relatively small, which makes difficult to specify major mutations reflecting the national specificity of FH. Moreover, the majority of studies accomplished so far did not explore possible associations of certain mutations with ethnic origin of patients. By now the only exception is the study of Karelian population showing the absence of typical Finnish mutations in the region that borders on Finland. It can be concluded that the important primary research partly characterizing the mutation spectrum in FH patients both in the European and Siberian parts of Russia has been done. However, it seems likely that the most interesting and comprehensive genetic studies of FH in Russia, concerning various mutations in different genes and the variety of ethnic groups in this multi-national country, are still to be undertaken.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3 protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Looking for a partner: ceruloplasmin in protein-protein interactions.

Ceruloplasmin (CP) is a mammalian blood plasma ferroxidase. More than 95% of the copper found in plasma is carried by this protein, which is a member of the multicopper oxidase family. Proteins from this group are able to oxidize substrates through the transfer of four electrons to oxygen. The essential role of CP in iron metabolism in humans is particularly evident in the case of loss-of-function mutations in the CP gene resulting in a neurodegenerative syndrome known as aceruloplasminaemia. However, the functions of CP are not limited to the oxidation of ferrous iron to ferric iron, which allows loading of the ferric iron into transferrin and prevents the deleterious reactions of Fenton chemistry. In recent years, a number of novel CP functions have been reported, and many of these functions depend on the ability of CP to form stable complexes with a number of proteins.

Role of arginine deiminase in thymic atrophy during experimental Streptococcus pyogenes infection.

Expression of gene of arginine deiminase (AD) allows adaptation of Streptococcus pyogenes to adverse environmental conditions. AD activity can lead to L-arginine deficiency in the host cells' microenvironment. Bioavailability of L-arginine is an important factor regulating the functions of the immune cells in mammals. By introducing a mutation into S pyogenes M46-16, we obtained a strain with inactivated arcA/sagp gene (M49-16 delArcA), deficient in AD. This allowed elucidating the function of AD in pathogenesis of streptococcal infection. The virulence of the parental and mutant strains was examined in a murine model of subcutaneous streptococcal infection. L-arginine concentration in the plasma of mice infected with S pyogenes M49-16 delArcA remained unchanged in course of the entire experiment. At the same time mice infected with S pyogenes M49-16 demonstrated gradual diminution of L-arginine concentration in the blood plasma, which might be due to the activity of streptococcal AD. Mice infected with S pyogenes M49-16 delArcA demonstrated less intensive bacterial growth in the primary foci and less pronounced bacterial dissemination as compared with animals infected with the parental strain S pyogenes M46-16. Similarly, thymus involution, alterations in apoptosis, thymocyte subsets and Treg cells differentiation were less pronounced in mice infected with S pyogenes M49-16 delArcA than in those infected with the parental strain. The results obtained showed that S pyogenes M49-16 delArcA, unable to produce AD, had reduced virulence in comparison with the parental S pyogenes M49-16 strain. AD is an important factor for the realization of the pathogenic potential of streptococci.

Neutrophil activation in response to monomeric myeloperoxidase.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Enzymatic and bactericidal activity of myeloperoxidase in conditions of halogenative stress.

Myeloperoxidase (MPO), found mainly in neutrophils, is released in inflammation. MPO produces reactive halogen species (RHS), which are bactericidal agents. However, RHS overproduction, i.e., halogenative stress, can also damage host biomolecules, and MPO itself may be targeted by RHS. Therefore, we examined the susceptibility of MPO to inactivation by its primary products (HOCl, HOBr, HOSCN) and secondary products such as taurine monochloramine (TauCl) and taurine monobromamine (TauBr). MPO was dose-dependently inhibited up to complete inactivity by treatment with HOCl or HOBr. TauBr diminished the activity but did not eliminate it. TauCl had no effect. MPO became inactivated when producing HOCl or HOBr but not HOSCN. Taurine protected MPO against inactivation when MPO was catalyzing oxidation of Cl- to HOCl, whereas taurine failed to prevent inactivation when MPO was working with Br-, either alone or in combination with Cl-. SCN- interfered with HOCl-mediated MPO inhibition. UV-vis spectra showed that heme degradation is involved in HOCl- and HOBr-mediated MPO inactivation. A negative linear correlation between the remaining chlorinating activity of HOCl- or HOBr-modified MPO and Escherichia coli survival upon incubation with MPO/H2O2/Cl- was found. This study elucidated the possibility of MPO downregulation by MPO-derived RHS, which could counteract halogenative stress.

N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride: Synthesis, interaction with DNA and evaluation of transfection efficiency.

А novel cationic chitosan derivative, N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), with different degrees of substitution (DS) was synthesized by a chemoselective interaction of 4-formyl-N,N,N-trimethylanilinium iodide with chitosan amino groups using a reductive amination method. Several factors (pH, reactant ratio, reaction time, and chitosan structure) were studied for their effects on the DS of the resulting TMAB-CS. The obtained derivatives were characterized by 1H NMR and FTIR spectroscopy. Turbidimetric titration showed enhanced solubility over a wide pH range even for low-substituted TMAB-CS. TMAB-CS provided strong DS-dependent binding of plasmid DNA. Dynamic light scattering measurements revealed the formation of stable polyplexes with hydrodynamic diameters of 200-300nm and ζ-potential of 20-30mV. TMAB-CS with relatively low DS (25%) demonstrated more pronounced transfection efficiency (up to 2000 cell/cm2) of plasmid DNA into the HEK293 cell line promoted by free TMAB-CS. The positive effects of lower DS can be related to a better polyplex dissociation within the cell. The cytotoxicity of TMAB-CS was comparable to that of the initial chitosan at concentrations up to 300ng/µL, even at high DS.

Functional link between ferroxidase activity of ceruloplasmin and protective effect of apo-lactoferrin: studying rats kept on a silver chloride diet.

Strongly pronounced argyrosis caused by adding AgCl to the feed of laboratory rats efficiently mimics the deficiency of ceruloplasmin (CP) ferroxidase activity. Bringing the concentration of AgCl in the feedstuff of lactating rats to 250 mg % and keeping their progeny (Ag-rats) for 3 months on the same silver-containing feed provided the serum iron content 1.4 times lower than that in the control group. Besides, the ferroxidase activity of CP dropped to zero. In CP purified from sera of Ag-rats two copper ions were substituted with two silver ions. Using rat models of both post-hemorrhagic and hemolytic anemia we showed that the deficiency of CP ferroxidase activity in Ag-rats affects the iron content in serum, though does not prevent the recovery of hemoglobin level accompanied by exhaustion of iron caches in liver and spleen. When apo-lactoferrin (apo-LF) was administered to Ag-rats suffering from either post-hemorrhagic or hemolytic anemia, both hemoglobin and serum iron were restored more rapidly than in the control animals. In independent experiments Ag-rats were compared with those fed on regular diet and the former displayed a prolonged 3-day stabilization of hypoxia-inducible factors 1 and 2 alpha (HIF-1a and HIF-2a) along with an increased serum concentration of erythropoietin. Introduction to Ag-rats of active CP separately or together with apo-LF reduced that effect to 1 day only. It is concluded that saturation of apo-LF with iron, provided by active CP, can strongly affect its protective capacity.

Ceruloplasmin decreases respiratory burst reaction during pregnancy.

Testing of pregnant women reveals weakening of neutrophil-mediated effector functions, such as reactive oxygen species generation. This study provides data confirming the phenomenon, gained through application of the flow cytometry technique. Key factors influencing neutrophil functional activity in blood plasma of pregnant women have not been detected so far. At the same time, concentration of ceruloplasmin - a copper-containing glycoprotein - is known to increase in blood significantly during pregnancy. We observed the negative correlation between ceruloplasmin concentration in blood plasma of pregnant women and the intensity of respiratory burst of neutrophils. Fractionation of plasma using gel-filtration revealed that ceruloplasmin-containing fraction demonstrated suppression of the respiratory burst reaction. Partial elimination of ceruloplasmin from the blood of pregnant women, performed with the help of specific antibodies and followed by immunoprecipitation, leads to an increased respiratory burst reaction. On the contrary, addition of ceruloplasmin to blood samples of healthy donors noticeably decreases the respiratory burst reaction. The results presented prove that change in ceruloplasmin level in plasma is necessary and sufficient for modulating the ability of neutrophils to produce reactive oxygen species during pregnancy.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.