Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

Evaluation of the kinematic parameters of normal-paced gait in subjects with gonarthrosis and the influence of gonarthrosis on the function of the ankle joint and hip joint

The aim of the study was to assess the variability of parameters characterising the gait of persons suffering from degenerative changes of the knee joint and their influence on the ankle and hip joints. The values of the angular changes in the knee, ankle and hip joints in the three planes of motion were assessed. Locomotion tests were performed on 27 persons, aged between 60 and 74, using Vicon 250, the three-dimensional analysis system. The sharpest deviations from the results of the control group were revealed in the transverse and frontal planes. Degenerative knee joint disease has changed the gait stereotype causing a reduction in the economy of gonarthrosis patients' locomotion, the influence of the disease on the function of the neighbouring joints is also distinctly marked.     

The extract from hop cones in plasma protects against changes following exposure to peroxynitrite

Humulus lupulus (Cannabaceae) is well known throughout the world as a raw material in the brewing industry. The antioxidative action of hop cones is poorly understood, therefore the aim of our present study was to investigate in vitro changes in human plasma induced by peroxynitrite in the presence of the highly purified extract from hop cones (Humulus lupulus). The aim of our study was also to explain the effect of the extract from hop cones on coagulation activity of human plasma treated with peroxynitrite. The action of the extract from hop cones was compared with the properties of a well-characterized commercial monomeric polyphenol — resveratrol (3,4′,5-trihydroxystilbene). The tested plant extract, like resveratrol, significantly inhibited protein carbonylation and nitration in plasma treated with ONOO⁻(0.1 mM). The extract from hop cones, like resveratrol, also caused a distinct reduction of plasma lipid peroxidation induced by ONOO⁻. Moreover, the tested extract modulated the coagulation properties of plasma treated with peroxynitrite. It seems that antioxidative activities of the highly purified extract from hop cones may be responsible for its medicinal properties.     

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

New class of quaternary ammonium salts, derivatives of methyl D-glucopyranosides

Reactions of two aromatic and two aliphatic amines with methyl 6-O-p-toluenesulfonyl-alpha-D-glucopyranoside or methyl 6-O-p-toluenesulfonyl-beta-D-glucopyranoside were performed on a micro-scale. The synthesis and preparative isolation methods have been developed for quaternary N-(methyl 2,3,4-tri-O-acetyl-6-deoxy-alpha- and -beta-D-glucopyranoside-6-yl)ammonium salts derived from three amines: trimethylamine, 2-methylpyridine, and pyridine. The reaction products were examined with 1H, 13C NMR spectroscopy. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-beta-d-glucopyranoside-6-yl)trimethylammonium tosylate was additionally analyzed with X-ray crystallography.     

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.     

Synthetic peptides mimicking antigenic epitope of Helicobacter pylori urease

Short peptides resembling the Helicobacter pylori urease antigen (UreB F8 Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe) with deleted aspartic acid and glutamic acid residues, anchored through a triazine linker via the N-terminal moiety to cellulose plate were prepared. The peptides were used for binding of antibodies from sera of patients with medically confirmed atherosclerosis. Recognition of the peptides was also tested with anti-Jack beans urease antibodies. The important role of a Gly-Gly spacer separating the peptides from the cellulose support was shown. Different patterns of binding of antibodies from H. pylori infected patients and anti-Jack bean urease antibodies were observed only in the case of pentapeptides. The peptide Gly-Gly-Leu-Val-Phe-Lys-Thr was recognized by most of the tested sera.