Nanoparticle-Modified Apoferritin Nanotransfer for Targeted Cytostatic Transport.

BACKGROUND: Ferritin is a globular intracellular protein that acts as the main reservoir for iron. Malignancies are associated with increased plasma ferritin concentrations. A number of studies show that tumor cells express high levels of transferrin receptors (TfR). Increased TfR expression was observed in prostate carcinoma. Apoferritin (APO) can be used as a protein nanotransporter into which a suitable medicinal substance can be encapsulated. Nanoparticles increase the permeability of tumor cells to nanotransporters and have a photothermal effect. The aim of this study was to encapsulate doxorubicin (DOX) into APO and to modify the resulting APO/DOX with gold (AuNPs) and silver nanoparticles prepared by green synthesis (AgNPsGS). METHODS: APO was characterized using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) - 120 V, 60 min, 24 mM Tris, 0.2 M glycine, 3 mM SDS. DOX fluorescence (Ex 480 nm; Em 650 nm) was observed, with a typical absorption maximum at 560 nm. Electrochemical measurement was performed in Brdicka solution (three-electrode setup). AgNPsGS were prepared by green synthesis using clover (Trifolium pratense L.). RESULTS: An electrophoretic study of APO and APO/DOX (5-100 μg/mL) was performed and the behavior of APO and APO/DOX (10 μM) as a function of pH was monitored. In an acidic environment, APO forms subunits of about 20 kDa; in an alkaline medium, it forms a globular protein of about 450 kDa. A change in APO/DOX mobility (about by 10%) was observed. A film of gold nanoparticles was applied to the APO/DOX surface. APO/DOX-AuNPs were washed with ultra-pure water. pH-dependent release of DOX a was monitored. The amount of DOX analyzed was increased by up to 50%. Furthermore, an AgNPsGS-DOX complex (1 mg AgNPsGS/100 μM DOX) was generated and prepared. Subsequently, the AgNPsGS-DOX complex was encapsulated into APO. To further improve therapeutic efficacy, the APO/AgNPsGS-DOX complex was coated with an Au layer. APO/AgNPsGS-DOX/AuNPs were stable and DOX was released from the complex after physical parameters had changed. CONCLUSION: APO nanocomplexes were prepared and modified to increase therapeutic efficacy against tumors. Tumor cell targeting was achieved by binding to TfR and via increased tumor cell permeability and retention. Release of the drug was made possible due to a pH change and photothermal activation that will now be tested. This work was supported by COST European Cholangiocarcinoma Network CA18122 and International Collaboration Project of The European Technology Platform for Nanomedicine. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 3. 2019 Accepted: 14. 5. 2019.

The effect of zinc and/or vitamin E supplementation on biochemical parameters of selenium-overdosed rats.

The normotensive (Wistar) and spontaneously hypertensive (SHR) rats were examined to assess the response of the organism to selenium (Se) overdose. Moreover, the effect of zinc (Zn) and vitamin E, i.e. dietary components interacting in many biochemical processes with Se, on the Se uptake was evaluated. The control group was fed an untreated diet, and the diets of two other groups were overdosed with Se in the form of sodium selenite (9 mg/kg) and supplemented with Zn (13 mg/kg). Two experimental groups were fed a diet supplemented with Zn (13 mg/kg) and Se at an adequate level (0.009 mg/kg); a half of the animals was supplemented with vitamin E. The results showed significant differences in the Se contents between the rat strains in case of Se-overdosed groups, where in the liver and kidney tissue Se contents of SHR rats exceeded 3- and 7-fold the normotensive ones. The Se uptake was altered by the vitamin E; no effect of Zn was observed. Activities of antioxidant enzymes were determined in the animal tissues indicating different patterns according to rat strain, tissue analysed, and administered Se dose. Thus, Se overdose, for instance, via an incorrectly prepared dietary supplement, can result in serious imbalances of the biochemical status of the animals.

Visfatin is actively secreted in vitro from U-937 macrophages, but only passively released from 3T3-L1 adipocytes and HepG2 hepatocytes.

Visfatin is a multi-functional molecule that can act intracellularly and extracellularly as an adipokine, cytokine and enzyme. One of the main questions concerning visfatin is the mechanism of its secretion; whether, how and from which cells visfatin is released. The objective of this in vitro study was to observe the active secretion of visfatin from 3T3-L1 preadipocytes and adipocytes, HepG2 hepatocytes, U-937, THP-1 and HL-60 monocytes and macrophages. The amount of visfatin in media and cell lysate was always related to the intracellular enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to exclude the passive release of visfatin. Visfatin was not found in media of 3T3-L1 preadipocytes. In media of 3T3-L1 adipocytes and HepG2 hepatocytes, the ratio of visfatin to the amount of GAPDH was identical to cell lysates. Hence, it is likely that these cells do not actively secrete visfatin in a significant manner. However, we found that significant producers of visfatin are differentiated macrophages and that the amount of secreted visfatin depends on used cell line and it is affected by the mode of differentiation. Results show that 3T3-L1 adipocytes and HepG2 hepatocytes released visfatin only passively during the cell death. U-937 macrophages secrete visfatin in the greatest level from all of the tested cell lines.

Muscular dystrophies and myopathies: the spectrum of mutated genes in the Czech Republic.

Inherited neuromuscular disorder (NMD) is a wide term covering different genetic disorders affecting muscles, nerves, and neuromuscular junctions. Genetic and clinical heterogeneity is the main drawback in a routine gene-by-gene diagnostics. We present Czech NMD patients with a genetic cause identified using targeted next-generation sequencing (NGS) and the spectrum of these causes. Overall 167 unrelated patients presenting NMD falling into categories of muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, and other myopathies were tested by targeted NGS of 42 known NMD-related genes. Pathogenic or probably pathogenic sequence changes were identified in 79 patients (47.3%). In total, 37 novel and 51 known disease-causing variants were detected in 23 genes. In addition, variants of uncertain significance were suspected in 7 cases (4.2%), and in 81 cases (48.5%) sequence changes associated with NMD were not found. Our results strongly indicate that for molecular diagnostics of heterogeneous disorders such as NMDs, targeted panel testing has a high-clinical yield and should therefore be the preferred first-tier approach. Further, we show that in the genetic diagnostic practice of NMDs, it is necessary to take into account different types of inheritance including the occurrence of an autosomal recessive disorder in two generations of one family.

Immunodominant semen proteins II: contribution of seminal proteins to female immune infertility.

Seminal fluid is a protective medium for sperm, but it also represents potential immunogenic structures for the female immune system. Anti-seminal antibodies may threaten early fertilization. The aim of our work is to detect and identify seminal proteins that are related to female isoimmunization. In this report, we quantified serum anti-seminal IgG antibodies. Seminal proteins were analysed by two-dimensional gel electrophoresis followed by immunoblotting. To identify IgG-binding proteins of interest, a proteomic approach was selected. The dominant seminal antigens were detected within the relative molecular mass ranging from 25 to 85 kDa and the isoelectric point from 5 to 7. The detected proteins were further identified as prostate-specific antigen, prostatic acid phosphatase, zinc-α-2-glycoprotein and zinc finger protein 778. Since these proteins were recognized by IgGs produced by infertile women and not by fertile women, we presume that major seminal antigens may play an important role in the pathogenesis of female immune infertility. Our study suggests the pattern of seminal proteins for further therapeutic attempts in the diagnosis of female immune infertility.

Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

Female serum immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens.

One in five couples of reproductive age has been diagnosed with infertility. Some diagnoses indicate an immunological basis for this disorder. Female immune infertility may be caused by iso-immunization by seminal components. We focused on the characterization of seminal proteins to illustrate the IgG, IgA and IgE immune responses of 31 infertile women. The biochemical characterization was performed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing, both of which were followed by immunoblotting analyses. IgG mainly recognized the antigens with relative molecular masses (Mr) 95 and 183 kDa and isoelectric points ranging from 6.9 to 7.0. The immunodominant antigens recognized by IgA had the Mr of 35 kDa and isoelectric points ranging from 6.2 to 7.2. The reactivity of IgE was not confirmed within our group of patients. The seminal IgG- and IgA -binding patterns were analysed immunochemically to determine the characteristics of possible seminal proteins associated with female immune infertility.

Autocrine effects of visfatin on hepatocyte sensitivity to insulin action.

Visfatin was originally described as an adipokine with insulin mimetic effects. Recently, it was found that visfatin is identical with the Nampt (nicotinamide phosphoribosyltransferase) gene that codes for an intra- and extracellular NAD biosynthetic enzyme and is predominantly expressed outside the adipose tissue. In the current study, we found strong protein and mRNA expression of visfatin in rat heart, liver, kidney, and muscle, while the expression of visfatin in visceral fat was significantly lower and undetectable in subcutaneous fat. The insulin-mimetic effects of visfatin (extracellular form of Nampt or eNampt) are controversial and even less is known about autocrine effects of visfatin (intracellular form of Nampt or iNampt). Since liver plays a major role in glucose metabolism, we studied visfatin effects on insulin-stimulated cellular glucose uptake in Fao rat hepatocytes using RNA interference (RNAi). RNAi-mediated downregulation of visfatin expression in Fao cells was associated with significantly reduced NAD biosynthesis (0.3+/-0.01 vs. 0.5+/-0.01 mmol/h/g, P<0.05) and with significantly decreased incremental glucose uptake after stimulation with insulin when compared to controls with normal expression of visfatin (0.6+/-0.2 vs. 2.2+/-0.5 nnmol/g/2 h, P=0.02). These results provide evidence that visfatin exhibits important autocrine effects on sensitivity of liver cells to insulin action possibly through its effects on NAD biosynthesis.

CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes.

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.

[Age-related post-translational modification of proteins in human tissue and its use determination of age in forensic medicine. Review]. / Závislost nekterých posttranslacních modifikací proteinu lidských tkání na veku a jejich pouzití pro urcování veku v soudním lékarství. Prehled poznatku.

This article deals with some of posttranslational modification of proteins which are, or could be useful to work out objective and standard methods for age estimation in forensic medicine. From many posttranslational modifications other than racemization, the evaluation of pentosidine, one of the products of nonenzymatic glycosylation, seems to be promising. The enlargement of menu of methods for age estimation is important mainly for forensic sciences when determination of the age of an unknown dead body is necessary. Morphological methods are quite often subjective and charged with errors.

Determination of saccharides in fruit juices by capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A fast method for the detection of cheap sweeteners is presented. Detecting the adulteration of foods rich in carbohydrates is complicated by the presence of variety of commercial sweeteners that are designed to match exactly the major carbohydrate profiles of these foods. Electrophoretic and mass spectrometric assays for the determination of fruit juice authenticity were developed. Capillary zone electrophoresis with indirect detection was employed to detect adulteration of juices demonstrated by the ratio of the concentrations of major low molecular mass saccharides (glucose, fructose and sucrose). Traces of oligosaccharides, which are not present in the sugar profiles of citrus fruits but are present in inexpensive sweeteners, were evaluated as the other group of target compounds. The fast determination of oligomeric starch hydrolysates in a complex matrix was tested by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and applied to orange juice. MALDI-TOFMS was shown to be a suitable method for the identification of adulteration of fruit juices by starch hydrolysates. The effects of the presence of salts and low molecular mass saccharides on the detection of oligosaccharides by MALDI-TOFMS were studied. Low molecular mass saccharides and organic acids decrease the detectability of oligosaccharides by MALDI-TOFMS, but the concentration of maltooligosaccharides present in juices sweetened with starch hydrolysates is high enough to be detected with good sensitivity.

Participation of complement in nasal polyposis.

No changes in immunoglobulin (IgG, IgM and IgA) and complement components C3 and C4 levels could be observed in patients suffering from both recurrent and acute nasal polyposis. However, in both groups of patients a significant decrease of the alternative pathway of complement activation (up to 80%) was found. In addition, patients suffering from the acute disease showed even decreased classical complement pathway (up to 20%). Examination of complement activation pathways was shown to be advantageous for the follow-up of the course of this disease.

Isolation, characterization and utilization of the fifth component (C5) of porcine complement.

The fifth component (C5) of porcine complement was isolated in immunochemically pure, homogeneous and native form. The isolated component was characterized, it has a molecular weight of 187,000 (under non-reducing conditions) and two non-identical chains of 112,000 and 76,000 under reducing conditions, respectively. The isolated C5 can be used for the detection of the C5b receptors on murine macrophages. Our findings are in good agreement with the results obtained using a rabbit and a human model, which suggests a high conservativeness of C5 during evolution. The described method of isolation of C5 from easily available porcine serum seems to be convenient for C5 receptors studies.

The expression of Fc and complement receptors in young, adult and aged mice.

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing.

Macrophages of athymic nude mice: Fc receptors, C receptors, phagocytic and pinocytic activities.

Expression of Fc receptors (FcR) for IgG1, IgG2A, IgG2B, IgM, IgA and IgE, binding of C3 and C5 complement components and phagocytic and pinocytic activities were determined in peritoneal and omental macrophages of nu/nu, nu/+ and +/+ Balb/c mice. nu/nu mice showed a higher proportion of FcR and complement receptor-bearing peritoneal macrophages along with a significantly higher phagocytic activity of peritoneal macrophages both in vitro and in vivo. Tests of pinocytic activity in these cells and phagocytic activity in omental phagocytes yielded similar results. We conclude that athymic mice compensate their immune defects by a higher phagocytic activity of their professional phagocytes and a higher expression of receptors mediating this process.

Characterization of the third component of pig complement and its utilization in a C3b receptor study.

The third component of the pig complement system (C3) was isolated in hemolytically active form and characterized. The C3 component is a beta-globulin with the molecular weight of 191,000 and is composed of 2 non-identical polypeptide chains of Mr 112,000 and 74,000. The isolated C3 can be used for the detection of the C3b receptor on the membranes of heterologous peritoneal macrophages.

Establishment of a macrophage hybrid population by the fusion of mouse peritoneal macrophages with thymoma cells BW5147.

A continuous macrophage hybrid population, designated MBW-1, was produced by the fusion of peritoneal macrophages from a normal AKR mouse with thymoma cells BW5147. The hybrid cells are resistant to HA selection medium. In their nearly triploid chromosome complement they carry metacentric chromosomes typical of BW5147 cells and express the typical macrophage-like characteristics (morphology of macrophages, adherence properties, phagocytosis, expression of macrophage-specific antigen and receptor for the C3b component of complement). However, they do not express Fc receptors and the receptor for SBA lectin. An interesting property is the high resistance to the toxic action of silica particles. Thanks to the unique properties, which were not observed in other, hitherto described macrophage-like cell lines, the MBW-1 cell population provides a useful material for study of macrophage functions.

C3b receptors on the cell membranes of the stimulated mouse macrophages. An immunohistochemical study.

C3b receptors were visualized on the cell membranes of stimulated mouse peritoneal macrophages (Mø) by the incubation with cross-reacting swine C3 with proof of this latter by FITC- or HRP-conjugated rabbit anti-swine-C3 antibody. The FITC-conjugate produced a granular and spotty fluorescence. In ultrastructure, the HRP-conjugate revealed minute dense areas of reaction product, whose modest numbers were seen in both aldehyde-prefixed and non-prefixed cell samples.