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Biomedical subjects

E C Veerman

Publications and source records attributed to E C Veerman.

At least 91 records · Page 5Linked to original sources
PubMed1989

Isolation of high molecular weight mucins from human whole saliva by ultracentrifugation.

A high molecular weight mucin fraction was prepared from human whole saliva using relatively mild conditions. The method involves ultracentrifugation of human whole saliva in the presence of 7.2 M urea and 0.5 M sodium chloride. The resulting preparation consists of a highly purified salivary mucin fraction, as judged by several purity criteria: molecular weight analysis of the final preparation by gel electrophoresis and analytical gel filtration indicated an apparent molecular weight greater than 10(6). Analytical isopycnic density centrifugation demonstrated that the preparation consisted of a mucin fraction with a buoyant density of approximately 1.47 g/ml. The final preparation comprised 12.8% protein, 31.8% N-acetylglucosamine, 11.5% N-acetylgalactosamine, 9.5% fucose, 21.4% galactose, 0.8% mannose, 10.3% N-acetylneuraminic acid, 1.7% sulphate and 0.16% fatty acids.

Centrifugation, Isopycnic↗
PubMed1989

Interaction of human salivary mucins with hydroxyapatite.

The interactions between hydroxyapatite (HAP) and several types of mucins (human whole salivary mucins, HWSM; ovine submandibular mucin, OSM; porcine gastric mucin, PGM) were compared using a quantitative assay. Of these mucins, HWSM displayed by far the highest binding to hydroxyapatite, followed by PGM and OSM, respectively. HWSM binding to hydroxyapatite was measured at pH 6.3 and 7.0. Data obtained appeared to fit empirically the Langmuirian adsorption isotherm. Apparent affinity constant (K-value) and maximum binding capacity (N-value), derived from these isotherms, indicated that upon lowering the pH the K-value increased from 0.7 to 0.8 ml/mg and the N-value from 2,050 to 2,640 micrograms/m2 hydroxyapatite. The pH-dependence of the HWSM-HAP interaction, measured over a large pH-range, at a fixed concentration of HWSM, indicated a two- to three-fold increase in binding upon lowering the pH from 7.5 to 5.5 At pH 5.5 the presence of HWSM induced an increased solubilization of hydroxyapatite, as deduced from the amounts of Ca2+ and phosphate-ions released. Addition of Ca2+ - and Mg2+ -ions (1mM) resulted in a decrease, both of HWSM binding and of the hydroxyapatite solubilization. These effects were more pronounced at the lower pH-values (pH less than 6.0).

Adsorption↗
PubMed1988

Influence of phytate on the adsorption of human salivary mucins onto hydroxyapatite.

The effect of phytate on the adsorption of purified human salivary mucins (HWSM) onto hydroxyapatite (HAP) was studied using three incubation conditions. a. Preadsorption of HWSM onto HAP for 24 h, followed by 4 h coadsorption with phytate, resulted in at most a 25% decrease in HWSM binding. b. Preincubation of HAP with phytate for 24 h, followed by 4 h coadsorption with HWSM, resulted in a 40% decrease in binding of HWSM to HAP. c. Simultaneous incubation of HWSM and phytate with HAP resulted in a 50% decrease in HWSM binding. In contrast, the adsorption of phytate to HAP was not affected, irrespective of the incubation set up used. The adsorption of phytate to hydroxyapatite was accompanied by an increase in the phosphate concentration of the solution. The molar ratio of phosphate solubilized/phytate bound to HAP was approximately 2 to 3. On the other hand, under conditions when all phytate added became bound to HAP (i.e. HAP in excess over phytate), no increase in Ca-ions was observed. However, when free phytate was present (phytate in excess over HAP) approximately one mole of Ca-ions was released per mole free phytate. For comparison, the effect of phytate on the adsorption of porcine gastric mucin (PGM) and ovine submandibular mucin (OSM) was studied. Under conditions when HWSM binding to HAP was decreased by 10%, binding of PGM and OSM decreased by 65% and 100%, respectively.

Adsorption↗
PubMed1987

SDS-PAGE analysis of the protein layers adsorbing in vivo and in vitro to bone substituting materials.

The composition of the protein layer adsorbed to the bone substituting materials, hydroxyapatite, beta-whitlockite, titanium and aluminium, in vivo (intramuscularly in guinea pig) and in vitro, was investigated using SDS-gel electrophoresis (SDS-PAGE). After in vivo implantation for 1 d mainly proteins with molecular weights between 10,000 and 20,000 were adsorbed. After 3 months the biolayer of the implanted biomaterials also contained proteins with molecular weights 35,000, 45,000, 60,000 and 200,000. No large qualitative differences in protein composition of the biolayers on the various implanted materials were found. In vitro incubation with human serum resulted in binding of proteins with estimated molecular weights of 30,000, 60,000 (albumin), 200,000 and greater than 200,000. It is suggested that the differences between in vivo and in vitro protein adsorption are due to proteolysis occurring in vivo in the vicinity of the implanted material.

Adsorption↗
PubMed1986

Immunohistological localization of factor VIII in placental endothelial cells.

DNA-recombinant studies have established that factor VIII-mRNA is present in liver and placental tissue. In a previous immunohistological study using monoclonal antibodies we have localized factor VIII in liver sinusoidal endothelial cells. In this paper we demonstrate that also the endothelial cells lining the fetal vessels in human placenta contain factor VIII. Based on the combined results of the present study and a previous study we favour the concept that factor VIII is synthesized in placental endothelial cells.

Antibodies, Monoclonal↗
PubMed1986

Localization of factor VIII-procoagulant antigen: an immunohistological survey of the human body using monoclonal antibodies.

Various organs, including liver, spleen, heart, lung, kidney, intestines, lymph nodes, pancreas, bone marrow, and thymus, were investigated for the presence of factor VIII-procoagulant antigen (VIIICAg) and factor VIII-related antigen (VIIIRAg), using a panel of monoclonal antibodies directed to factor VIII-von Willebrand factor in combination with a sensitive immunoperoxidase staining technique. In addition to hepatic sinusoidal endothelial cells, the presence of VIIICAg was demonstrated in mononuclear cells sporadically present in lymph nodes, in the alveolar septa of lung, and in the red pulp of spleen. The identity of these mononuclear cells could not be unequivocally determined. Based on morphological criteria, however, it is tentatively concluded that these cells are nonlymphoid and belong to the mononuclear phagocyte system. The presence of VIII-RAg was confined to vascular endothelial cells, hepatic sinusoidal endothelial cells, cells lining the venous sinuses of the red pulp of the spleen, cells lining renal glomeruli and lung capillaries, platelets, and megakaryocytes.

Antibodies, Monoclonal↗
PubMed1984

Temperature dependence of energy-transducing functions and inhibitor sensitivity in chloroplasts.

A comparative analysis of the temperature dependence of energy-transducing reactions in spinach (Spinacia oleracea) chloroplasts and their sensitivity for uncouplers and energy-transfer inhibitors at different temperatures is presented. Arrhenius plots reveal two groups of transitions, around 19 degrees C and around 12 degrees C. Activities that show transitions around 19 degrees C include linear electron flow from water to ferricyanide, its coupled photophosphorylation, the dark-release of the fluorescent probe atebrin, and the slow component of the 515 nm (carotenoid) absorbance decay after a flash. The transitions around 12 degrees C are observed with pyocyanine-mediated cyclic photophosphorylation, light- and dithioerythritol-activated ATP hydrolysis, the dark-release of protons, and the fast 515 nm decay component. It is suggested that both groups of temperature transitions are determined by proton displacements in different domains of the exposed thylakoid membranes. The effects of various uncouplers and an energy-transfer inhibitor are temperature dependent. Some uncouplers also show a different relative inhibition of proton uptake and ATP synthesis at lower temperatures. The efficiency of energy transduction (ATP/e(2)) varied with temperature and was optimal around 10 degrees C.

Journal Article↗
PubMed1984

Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium.

We have studied the role of factor VIII-von Willebrand factor (FVIII-vWF) in both platelet adherence to subendothelium and ristocetin-induced platelet aggregation using monoclonal antibodies to human FVIII-vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Antibodies, Monoclonal↗
PubMed1983

A rapid one-step immunoradiometric assay for factor VIII-procoagulant antigen utilizing monoclonal antibodies.

A two-site immunoradiometric assay for factor VIII-procoagulant antigen (VIIICAg) that relies completely on monoclonal antibodies has been developed. By selecting an appropriate combination of these antibodies, it was possible to develop an assay in which the radiolabelled monoclonal antibody did not inhibit the binding of antigen to the solid-phase monoclonal antibodies. Thus, the entire test could be carried out as a one-step procedure. With this one-step assay, an amount of 0.0025 U VIIICAg/ml plasma could be detected after 4 hr of incubation, whereas 18 hr of incubation resulted in a lower limit of sensitivity of 0.0005 U VIIICAg/ml. The use of a one-step assay provides a significant advantage over the conventional two-step assay by simplifying, shortening and rendering the performance of the assay more convenient.

Antibodies, Monoclonal↗
PubMed1982

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents.

The kinetics of the oxidation-reduction reactions of cytochrome c1 with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c1 by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c1 with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reactions rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c1 with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4 . 10(4), 3.2 . 10(3), 3.8 . 10(4) and 1.3 . 10(8) M-1 . s-1 (pH 7.9, I = 0, 10 degrees C), respectively. Application of the Debye-Hückel theory to the data of the ionic-strength-dependence studies of these redox reactions of cytochrome c1 yielded for ferrocytochrome c1 and ferricytochrome c1 a net charge of --5 and --4, respectively. The latter value is close to that of --3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c1 governs the reaction rate with small redox molecules.

Animals↗
PubMed1982

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis.

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.

Animals↗
PubMed1981

The ionic strength dependence of the rate of a reaction between a small ion and a large ion with a dipole moment.

The ionic strength dependence of the rate constant of a reaction between a small ion and a large ion with a dipole moment (e.g. a protein) is described. This description takes into account only the electrostatic interactions between the two ions. This approach agrees with the Marcus theory treatment of the electrostatic interactions and also with the Debye-Hückel theory which is based on changes in the activity coefficients of the reactants. The contribution of the dipole moment of the protein to the ionic strength dependence of the rate constant has been calculated. A method is described whereby one can calculated the charge of the protein without knowing the precise ionic strength dependence of the rate constant. Two applications are mentioned to illustrate the usefulness of the method.

Cytochrome c Group↗
PubMed1981

Ionic strength effects on cytochrome aa3 kinetics.

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa3 can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa3 increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa3 complex limits the ferrocytochrome c association to the low affinity site. 2. At low ionic strength, the reduction of cytochrome c-aa3 complex by ferrocytochrome c1 proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c1 and aa3. 3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa3 complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c. 4. Dissociation rates of both high and low affinity sites on cytochrome aa3 were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 microM for the high and low affinity site, respectively.

Animals↗
PubMed1980

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex.

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa3 and to the (porphyrin) cytochrome c-cytochrome aa3 complex. 2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2.10(8) M-1.s-1 and about 10(7) M-1.S-1 (I=8.8 mM, pH 7.0, 10 degrees C), respectively. 3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa3 reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa3. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa3 has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa3 has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.

Animals↗
PubMedDate not supplied

Detection of factor VIII/coagulant antigen in human liver tissue.

Factor VIII, a high molecular weight glycoprotein complex which has an important role in haemostasis, consists of two immunologically as well as functionally discernible moieties that can be isolated separately. These are factor VIII/von Willebrand factor (FVIII/vWF) which is associated with the factor VIII-related antigen (FVIIIRAg), and the factor VIII/procoagulant activity (FVIII/C) which is associated with the factor VIII/procoagulant antigen (FVIII/CAg). The FVIII/C activity is decreased or absent in patients with haemophilia A (for a review of the structure and function of the factor VIII complex, see refs 1 and 2). Immunological techniques, combined with cell culture, have demonstrated that FVIIIRAg is present in and synthesized by endothelial cells and megakaryocytes. However, the organ and/or cell type responsible for the production of FVIII/C has not been established. Indirect evidence derived from organ transplantation in experimental animals suggests that the liver is the most likely organ for FVIII/C production. Here we have used a monoclonal antibody against FVIII/CAg in combination with a sensitive immunostaining technique to demonstrate the presence of FVIII/CAg in hepatic sinusoidal endothelial cells.

Antibodies, Monoclonal↗