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INTRODUCTION: Pathological formation of blood vessels plays a key role in the growth and metastasis of tumors and also in several serious ophthalmological diseases such as wet age-related macular degeneration (AMD) or diabetic retinopathy. In AMD treatment, aflibercept (tradename EYLEA®) is used to deactivate the underlying pathological neovascularisation. Aflibercept is a recombinant fusion protein which binds to vascular endothelial growth factor (VEGF) receptors, thereby inhibiting VEGF pathway activation. VEGF is one of the most important angiogenesis factors. OBJECTIVE: This analysis investigates lasting efficacy of aflibercept in vitro for later application as therapeutic agent against macular degeneration (AMD). MATERIAL AND METHODS: VEGF-ELISA assays were performed to investigate binding affinities at different aflibercept concentrations. The impact of VEGF on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated using proliferation assays. Moreover, time-dependent kinetic studies were performed to analyze different aflibercept storage durations with regard to its inhibitory capabilities on human VEGF. RESULTS AND CONCLUSION: Our results reveal that aflibercept significantly lowers the amount of unbound VEGF as well as the proliferation rate of HUVEC. Moreover, in contrast to specifications given by the manufacturer, aflibercept retains its full inhibitory effect up to at least 120h after transference from the original vial into the injection syringe.

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A transistor pH electrode (ion sensitive field effect transistor), placed in the upper dentures of eleven xerostomia patients and five healthy volunteers, was used to register pH changes in five-, six- and seven-day-old dental plaque. A mouth rinse with a 10% sucrose solution caused a pH fall of about three decades. A significant difference in duration of critical plaque pH was observed: in xerostomia patients, a 10% longer period of pH less than 5.7 was registered during 60 min following a sucrose rinse. Normal oral functions were not influenced by the denture with an integrated electrode. This method is usable for plaque pH registration in xerostomia patients.

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A comparative study was performed on the influence of two similar tertiary amines, procaine and nicotinoyl-procaine, on isolated kidney and cerebellum cells in culture. Shortly after the addition of the test substances vesicles are found in the cells and the cells are more broadly attached to the underground. But the attachment of microfilaments is scarcely adhered. Daily addition of procaine or nicotinoyl-procaine, resp., to cell cultures leads to an increase of protein concentration and enzyme activities. Particularly organotypical enzymes are enhanced, as gamma-glutamyltransferase in kidney cells and creatine kinase in cerebellum cells.

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The comparative studies are continued on the influence of two tertiary amines, procaine and nicotinoyl-procaine, on isolated heart and skeletal muscle cells in culture. A daily addition of these substances to cell cultures produces an increase in the specific activities of glutamate dehydrogenase and glutamate-oxaloacetate transaminase and an inhibition of alteration in the isoenzyme pattern of lactate dehydrogenase. The presence of procaine or nicotinoyl-procaine, resp., inhibits thymidine incorporation by cells in culture, whereas the incorporation of thymidine is increased after pretreatment of cells with these substances. These results support the findings of an influence of said tertiary amines on the metabolism of cells in culture.

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Frog motor nerves and isolated heart cells from neonatal rats were incubated with solutions of open chain crown-type polyether or pyridinophane cryptand. The following alterations in membrane excitability and energy consumption were found: 1. The non-cyclic ligand stabilizes the resting potential of the frog nerve and reduces the pulsation rate of heart muscle cells. It is reversibly bound at the cell surface and does not affect the energy metabolism of the heart cells. (formula: see text) 2. The cryptand 1,12-dioxo-2,11-diaza-5,8,21,24-tetraoxa[12-8(2,11)] (2,6)-pyridinophane) ([2.2.1py]-diamide) is irreversibly bound by the tissues. It facilitates the depolarization of the nerve and shows a positively chronotropic effect upon the heart muscle cells. Single treatment of the cell cultures with 10 microgram [2.2.1py]-diamide per ml medium increased the activities of lactate dehydrogenase and of creatine kinase. When the cell cultures were treated three times at 24 h intervals with 10 microgram complexone/ml, the creatine kinase activity of the heart muscle cells decreased by about 40%. The physiological properties of the ligands are correlated with the stability of their alkali metal ion complexes and with the rate constants of complex formation. It is concluded that [2.2.1py]-diamide can act as a passive carrier for Na+ K+.

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A comparative study was performed on the influence of a series of chemically defined local anaesthetics such as procaine, nicotinoylprocaine and butacaine on isolated skeletal muscle cells and heart cells in culture. About 30 min after addition of local anaesthetics to cell cultures vesicles appear in the cells. These formations are reversible and need a minimum concentration of 13(-3) mol/l procaine, 10(-4) mol/l nicotinoylprocaine, or 10(-4) mol/l butacaine, respectively. Soon after addition of local anaesthetics a reduction of oxygen consumption is measurable in both cell types. The degree of this reduction depends on the chemical structure and the concentration of local anaesthetics and on the kind of cells. After daily addition of local anaesthetics to cell cultures metabolic parameters were studied. The local anaesthetics, except for butacaine, produce an increase of protein concentration, creatine kinase and lactate dehydrogenase activity per sample and an increase of the specific activity of lactate dehydrogenase. These increases depend also on the concentration of local anaesthetics and on the kind of cells. These results support the hypothesis of local anaesthetics action via membrane lipids.

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(Adenine-14C) or (gamma-32P)-labelled 2,2'[1-(9-adenyl)-1'-(tri-, diphosphoryl-oxymethyl]-dihydroxydiethylether (rroANP) was obtained from ANP by cleavage of the C-2--C-3' bond by sodium periodate oxidation and subsequent borohydride reduction. Binding of rroANP to rat liver mitochondria revealed carrier-linked (atractyloside-sensitive) and unspecific (atractyloside-insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as a competitive inhibitor for ANP uptake with Ki = 9.3 X 10(-5) M. Experimental rroANP confirmed that an intact adenine base and three anio.nic charges of the phosphate chain are essential for the recognition between ANP-carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier-nucleotide complex requires an intact ribofuranoside ring system.

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Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals 31.5 mM(-1) X cm(-1). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis. Liver phosphoglycerate kinase is inactivated when modified with either p-chloromercuribenzoate or 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2, sugessting the presence of a disulfide bridge. Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000-50000. By analytical dodecylsulfate-polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results in the value 37500 obtained by thin-layer gel chromatography in 6 M guanidine-HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w equals 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v 0.747 ml x g(-1). The diffusion coefficient was found to be 76.9 mum2 x s(-1) by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine-liver phosphoglycerate kinase are: frictional ratio f/f0 equals 1.18, axial ratio equals 3.3, maximal degree of hydration equals 0.1 g per g of protein. Bovine-layer phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine-HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme. Some characteristics of bovine-liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.

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