Anti-infective DNase I coatings on polydopamine functionalized titanium surfaces by alternating current electrophoretic deposition.

Implant-associated infections (IAIs) can cause serious problems due to the difficult-to-treat nature of the biofilms formed on the implant surface. In mature biofilms, the matrix, which consists of polysaccharides, proteins, lipids and extracellular DNA (eDNA), forms a protective environment for the residing bacteria, shielding them from antibiotics and host defenses. Recently, the indirect prevention of biofilm growth through the degradation of eDNA using an enzyme, such as deoxyribonuclease (DNase) I, has gained attention and is regarded as a promising strategy in the battle against IAIs. In this study, coatings of DNase I were applied on titanium implant materials and their anti-infective properties were investigated. First, the effectiveness of alternating current electrophoretic deposition (AC-EPD) as a novel processing route to apply DNase I on titanium was examined and compared with the commonly applied diffusion methodology (i.e. classic dipping). For the same processing time, the use of AC-EPD in combination with a polydopamine (PDA) coupling chemistry on the titanium electrode surface significantly increased the protein deposition yield as compared to classic dipping, thereby yielding homogeneous coatings with a thickness of 12.8 nm and an average surface roughness, Sa, of ∼20 nm. X-ray photoelectron spectroscopy confirmed the presence of peptide bonds on all DNase-coated substrates. Time-of-flight secondary ion mass spectrometry detected a more dense DNase I layer in the case of AC-EPD for electrodes coupled as anode during the high-amplitude half cycle of the AC signal. The enzyme activity, release kinetics, and shelf life of DNase I coatings were monitored in real-time using a quantitative qDNase assay. The activity of DNase I coatings produced using AC-EPD was three time higher than for coatings prepared by classic dipping. For both deposition methods, a high initial burst release was observed within the first 2 h, while some activity was still retained at the surface after 7 days. This can be explained by the stable attachment of a small fraction of DNase to the surface through covalent bonding to the PDA layer, while superimposing DNase deposits were only loosely bound and therefore released rapidly upon immersion in the medium. Interestingly, coatings prepared with AC-EPD exhibited a prolonged, gradual release of DNase activity. The AC-EPD DNase coatings significantly reduced biofilm formation of both Staphylococcus epidermidis and Pseudomonas aeruginosa up to 20 h, whereas DNase coatings prepared by short classic dipping only reduce S. epidermidis biofilm formation, and this to a lesser extent as compared to AC-EPD DNase coatings. Overall, this study indicates that AC-EPD allows to rapidly concentrate DNase I on PDA-functionalized titanium, while maintaining the enzyme activity and anti-infective ability. This highlights the potential of AC-EPD as a time-efficient coating strategy (as opposed to the much slower dip-coating methodologies) for bioactive molecules in a wide variety of biomedical applications.

Influence of fatty acid diets on gene expression in rat mammary epithelial cells.

BACKGROUND: This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty. METHODS: Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets. RESULTS: Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. CONCLUSION: Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model.

[Initial experience with selection procedures for admission to medical school]. / De eerste ervaringen met selectie voor de artsopleiding.

Admittance to a medical school in the Netherlands has for decades been based on a grade point average weighted lottery system of secondary school leavers. Since 2000, the Dutch Higher Education and Scientific Research Act has given medical schools the option of selecting candidates. In 2000, two of the eight Dutch medical schools started selection experiments for 10 percent of their places. Leiden University Medical Center invited school leavers who had studied a more varied range of extra subjects to attend a 10-day summer school. All 54 candidates were ranked on the basis of assessments and tests; 24 of them were admitted. Utrecht University invited students with a higher education degree to a selection day. An application form, a structured interview and a questionnaire determined the ranking of 53 candidates; 24 of them were admitted. Both schools were satisfied with the manner in which the selection procedure worked. However, it is not yet possible to draw any definite conclusions about the effectiveness of the selection procedure.

[Ex situ sleeve resection and autotransplantation of the left lower pulmonary lobe after extended pneumonectomy]. / Ex-situ-Manschettenresektion mit Autotransplantation des linken Lungenunterlappens nach erweiterter Pneumonektomie.

A 69-year-old patient with reduced pulmonary function was diagnosed as suffering from non-small cell lung cancer of the left lung invading the main bronchus, pulmonary artery and left atrium. Staging examinations were negative. Using cardiopulmonary bypass, an extended pneumonectomy with partial resection of the left atrium was performed. The cardiac defect was closed with a pericardial patch. The lower lobe was divided ex situ from the upper lobe and reimplanted with anastomosis of the lower pulmonary vein to the left auricle. After a totally uneventful course the patient is in good condition and free of tumor recurrence 2.5 years postoperatively.

Physiologic consequences of pneumonectomy. Consequences on the esophageal function.

Esophageal and upper gastrointestinal dysmotility occur after both pneumonectomy without pulmonary replacement and recipient pneumonectomy for thoracic organ transplantation. After pneumonectomy without pulmonary replacement, there is a shift of the esophagus to the side of pneumonectomy and disturbance of esophageal peristalis. After recipient pneumonectomy for thoracic organ transplantation, esophageal dysmotility and delayed gastric emptying are common. Injury of the vagal nerves, local ischemia, postoperative scarring of the esophagus and mediastinum, and disturbance of the autonomic nervous systems are the major causes of the abnormality. To reduce the incidence of esophageal dysmotility after pneumonectomy, every effort should be made during surgery to prevent direct injury of the esophagus or the vagal nerves.

The delta-subunit of pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus is a redox-active, iron-sulfur protein: evidence for an ancestral relationship with 8Fe-type ferredoxins.

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.

A chromogenic test to determine the procoagulant phospholipids in platelet-rich plasma and whole blood.

We have developed a chromogenic assay to measure the phospholipid-related procoagulant activity (PPA) in whole blood, or platelet-rich plasma. The test is based upon thrombin formation from prothrombin by prothrombinase and is designed in such a way that procoagulant lipids are rate limiting for the prothrombinase activity. In the chromogenic test PPA concentrations equivalent to 0-10 nM phospholipid vesicles containing 75% phosphatidyl choline (PC) and 25% phosphatidyl serine (PS) can be measured. The thrombin, which develops during the test, is measured with a chromogenic substrate. By the action of thrombin on this chromogenic substrate p-nitroaniline is liberated, which causes an increase in absorbance. Thrombin formed in the assay mixture activates the present platelets. This causes a linear increase of the velocity of thrombin generation during the test, i.e. a parabolic increase of product formation. For that reason the thrombin generation in time is characterized by two parameters, the basal PPA (PPA-B) of the original mixture and the increase in PPA due to platelet activation (PPA-A). To determine these figures the absorbency-data were fitted to parabolas. In most cases the contribution of PPA-A to the total amount of formed thrombin becomes considerable already after 30 s. Preliminary tests show that PPA-B activity in whole blood or platelet-rich plasma of patients with a thrombotic disorder is significantly higher than the activity of a control group of the same age.

Development of a rapid and sensitive chromogenic heparin assay for clinical use.

A new sensitive chromogenic heparin assay is developed, which is well suited for clinical use. For the assay two reaction mixtures are required which can be lyophilized and reconstituted on the day of use. These reagents are stable during at least 6 h. Only two time-dependent pipetting steps are necessary. Any compound that inactivates thrombin, or can potentiate thrombin inactivation by an inhibitor, can be measured with this assay, including standard heparin, low molecular weight heparins, hirudin, alpha-NAPAP, pentosan polysulphate and dermatan sulphate. It is shown that heparin can be measured accurately in whole blood and in plasma. By addition of dextran sulphate to one of the reagents a platelet factor 4-insensitive assay is developed, so heparin can be measured even in blood that is partially activated and thus contains platelet factor 4 which neutralizes heparin.

Development of a sensitive and rapid chromogenic factor IX assay for clinical use.

A chromogenic factor IX assay is developed which requires only two time-dependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XIa, thrombin, CaCl2, and phospholipids. Then factor XIa activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXa, factor VIIIa, Ca ions, and phospholipids) rapidly activates factor X. Finally, ethylenediaminetetraacetic acid plus a chromogenic substrate are added to stop the reaction and to measure formed factor Xa. Factor Xa formation is proportional to the plasma factor IX concentration (from 0 to 140%). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 degrees C. A new isolation procedure for factor VIII is described. Factor VIII is purified from bovine plasma in a few steps with a yield of 20% and a 8,000-fold purification.

Development of a simple chromogenic factor VIII assay for clinical use.

The aim of this study was the development of a simple chromogenic factor VIII assay for practical clinical use. The criteria that the assay fulfils are: (1) The method is so sensitive that even 1% factor VIII in human plasma is easily detected. (2) The method is linear in the amount of factor VIII from 0 to 200% in plasma. (3) The pipetting scheme is very simple; two reagents are prepared, reagent 1 (factor IXa, thrombin, Ca2+ and phospholipids) and reagent 2 (factor X). Then we pipet at t = 0 s, 100 microliters diluted plasma + 100 microliters reagent 1 in a reaction tube; at t = 30 s, 100 microliters reagent 2 in the same tube and at t = 90 s, 200 microliters of the reaction mixture in a cuvette with 700 microliters EDTA buffer (stop buffer) and the formed factor Xa is measured with a chromogenic substrate. (4) The reaction components are stable during at least a whole working day. Factor VIII was measured in an assay using bovine clotting factors, so one avoids the risk of viral infections, which one might catch by working with clotting factors isolated from human plasma.

Localization of the inhibitory site(s) of pentosan polysulphate in blood coagulation.

We studied the inhibitory effect of pentosan polysulphate (PPS, Hémoclar) on thrombin formation in blood coagulation. In contrast to a current hypothesis the antithrombin III independent effect of PPS on blood coagulation is not caused by preventing the binding of the factors IX, IXa, X, Xa, VIII, V, Va and II onto procoagulant phospholipids. We investigated the activation by thrombin of factors I, V and VIII. A strong inhibitory effect of PPS on factor VIII activation could be observed. Inhibition of the activation of factor V to the same extent requires about 30-fold higher concentrations of PPS, whereas the activation (clotting) of fibrinogen is not inhibited. The effect of PPS on factor VIIIa is two-fold: A) it inhibits its formation and B) it inhibits its function probably by the formation of a factor VIIIa-PPS complex. Prothrombinase, constituted of purified factors Xa, Va and phospholipids was not inhibited by PPS, neither were incomplete forms of this enzyme, lacking phospholipids or factor Va. The complete factor X activating enzyme (factors IXa, VIIIa and phospholipids), however, was strongly inhibited, but incomplete forms, lacking factor VIII, were not. The inhibition of the complete enzyme can be explained by reversible binding of PPS to factor VIIIa (causing an inhibition of its function) and it is not an effect on the enzymatic function of the complete enzyme. On saturation of the enzyme with an excess of factor VIIIa no inhibition by PPS is noticed. We postulate therefore that the antithrombin III independent inhibitory effect of PPS on thrombin generation on blood coagulation is by interaction with factor VIIIa. This effect is additional to the heparin-like action of PPS, i.e. potentiation of the activity of antithrombin III and/or heparin cofactor II.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of histologic and quantitative techniques in evaluation of therapy for experimental Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia was induced in rats by the administration of corticosteroids, and histologic and quantitative techniques were compared in the evaluation of the severity of the disease and response to therapy. A highly significant correlation was found between the histologic score of the extent of alveolar involvement (the standard method of assessment) and the number of P. carinii cysts and nuclei in lung homogenates, lung weight, and lung weight/body weight ratio. Clear differences were noted between rats which responded well and rats which responded poorly to therapy by all techniques. Quantitation of P. carinii cysts and nuclei revealed a 10(4)-fold reduction in organism burden with successful treatment. Thus, these techniques should be helpful in the development of testing of new antimicrobial agents in the rat model of pneumocystosis.

Decarboxylated bone Gla-protein as a substrate for hepatic vitamin K-dependent carboxylase.

Bovine bone Gla-protein (B.G.P.) was prepared and decarboxylated into descarboxy-B.G.P. (d-B.G.P.). The latter was purified and identified as decarboxylated osteocalcin. Both crude and purified d-B.G.P. are good substrates for vitamin K-dependent carboxylase. Because the Km of this enzyme for d-B.G.P. is low, the latter is a better substrate than the frequently used pentapeptide FLEEL or exogenous protein substrates such as descarboxyprothrombin.

Activation of human prothrombin by stoichiometric levels of staphylocoagulase.

The activation of human prothrombin by the bacterial protein staphylocoagulase proceeds via the formation of a very stable equimolar complex. Unmasking of the active center in the prothrombin moiety of the complex is not caused by limited proteolysis. The kinetics of activation of human prothrombin by staphylocoagulase has been studied. The second order rate constant at pH 7.5, 37 degrees C, is 3.3 X 10(6) M-1 S-1. This reaction rate is close to reported diffusion-controlled rates of protein-protein interaction. The dissociation constant of the complex was too low to be measurable. From the kinetic data it is assumed that the first order rate constant for dissociation is orders of magnitude less than 10(-5) S-1. However, dissociation of the complex did occur in the presence of sodium dodecyl sulfate. Equimolar amounts of staphylocoagulase protect human thrombin, but not human factor Xa and bovine thrombin, against inactivation by antithrombin III. From these findings we postulate that tertiary structural changes in the thrombin region of prothrombin caused by a highly specific interaction between staphylocoagulase and that region unmask the active site.

Vitamin K-dependent carboxylases from non-hepatic tissues.

The presence of vitamin K-dependent carboxylase was investigated in the microsomal fraction of 20 different types of bovine tissue. Except for muscle, veins, lymphocytes and bone membrane, carboxylase was found in all these preparations, albeit in varying amounts. No differences could be detected between these carboxylating systems with respect to their affinity for vitamin K and warfarin. Most of the endogenous substrates had some affinity towards antiprothrombin or antifactor X.

The power of transformation.

CONCLUSION: The answer to the question, how is man empowered to change? can be given in this form: We are empowered to change when we experience trust and acceptance coming to us from another person or group. Change occurs when our power of being is affirmed by other centers of power. This affirmation of our power of being overcomes the anxiety of nonbeing implied in every change. It supplies us with the courage to accept our acceptance and to exist in spite of threats to our existence. The content of trust in our experiencing that has taken the place of the prior anxiety in our experiencing reduces the disturbances that distorted our perception of reality. This enables us to receive reality more accurately and to symbolize it more exactly. Change then is an internal metamorphosis that is the result of a courage educed in us by external forces. The transformation of our internal experiencing is the prior condition and ground for the transformation of our symbolizations.

The ontological character of anxiety.

CONCLUSION: We conclude from our analysis of Tillichian and Rogerian anthropology that anxiety is ontological in character. It is ontological because it is the necessary concomitant condition of the fact and structures of existence. Man is faced with one basic threat-the possibility of non-being. In an absolute form, he is threatened with the total extinction of being. In an absolute form, he is threatened with the total extinction of being. In a relative form, he is threatened with personal death and with the failure of personal fullillment-with the loss of being and becoming. Ontological anxiety has to do with the absolute threat of extinction and ontic anxiety with the relative threat to self-preservation and self-enhancement. From this condition there is no escape. Refusal to accept the inescapable results in neurotic anxiety, a nonessential concomitant of being which, therefore, can be removed. These distinctions are revealed by an ontological and phenomenological analysis of human existence that brings ontology and psychology into a complementary relationship.