Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.     

Bioassay directed identification of natural aryl hydrocarbon-receptor agonists in marmalade

Citrus fruit and citrus fruit products, like grapefruit, lemon and marmalade were shown to contain aryl hydrocarbon receptor (AhR) agonists, as detected with the DR CALUX bioassay. This is of interest regarding the role of the Ah-receptor pathway in the adverse effects of dioxins, PCBs and other aromatic hydrocarbons. So far it is unclear which compounds in citrus fruit are responsible for the AhR-mediated activity and whether regular exposure to these compounds can cause effects comparable to, e.g. dioxins. The present study aimed at developing a method for identifying unknown Ah-receptor agonists in citrus products based on bioassay directed analysis, using marmalade as a first target. Following extraction with hexane and purification on an aluminium oxide-column, the extract was fractionated by HPLC using a C-18 semi-preparative column. Fractions were extracted, solvent-exchanged into dimethylsulfoxide and subsequently tested with DR CALUX. Extracts were shown to contain primarily coumarins, furocoumarins (FCs) and polymethoxyflavones (PMFs). Identification of fractions most active in the bioassay via LC/MS revealed that bergapten (an FC) is the most important Ah-receptor agonist in marmalade. The approach and method developed resulted in the successful identification of the bioactive component. However, potential pitfalls of the procedure will be discussed.     

Novel polynomial Bernstein bases and Bézier curves based on a general notion of polynomial blossoming

We introduce the G-blossom of a polynomial by altering the diagonal property of the classical blossom, replacing the identity function by arbitrary linear functions G=G(t). By invoking the G-blossom, we construct G-Bernstein bases and G-Bézier curves and study their algebraic and geometric properties. We show that the G-blossom provides the dual functionals for the G-Bernstein basis functions and we use this dual functional property to prove that G-Bernstein basis functions form a partition of unity and satisfy a Marsden identity. We also show that G-Bézier curves share several other properties with classical Bézier curves, including affine invariance, interpolation of end points, and recursive algorithms for evaluation and subdivision. We investigate the effect of the linear functions G on the shape of the corresponding G-Bézier curves, and we derive some necessary and sufficient conditions on the linear functions G which guarantee that the corresponding G-Bézier curves are of Pólya type and variation diminishing. Finally we prove that the control polygons generated by recursive subdivision converge to the original G-Bézier curve, and we derive the geometric rate of convergence of this algorithm.     

Eigenvalues of ‐Free Graphs and the Connectivity of Their Independence Complexes

Let G be a graph on n vertices, with maximal degree d, and not containing as an induced subgraph. We prove:

• 1.
• 2.

Here is the maximal eigenvalue of the Laplacian of G, is the independence complex of G, and denotes the topological connectivity of a complex plus 2. These results provide improved bounds for the existence of independent transversals in ‐free graphs.     

Formal Proofs for the Security of Signcryption

Signcryption is an asymmetric cryptographic method that provides simultaneously both message confidentiality and unforgeability at a low computational and communication overhead. In this paper we propose realistic security models for signcryption, which give the attacker power to choose both messages/signcryptexts as well as recipient/sender public keys when accessing the signcryption/unsigncryption oracles of attacked entities. We then show that Zheng's original signcryption scheme is secure in our confidentiality model relative to the Gap Diffie-Hellman problem and is secure in our unforgeability model relative to a Gap version of the discrete logarithm problem. All these results are shown in the random oracle model.     

Independent systems of representatives in weighted graphs

The following conjecture may have never been explicitly stated, but seems to have been floating around: if the vertex set of a graph with maximal degree Δ is partitioned into sets V _i of size 2Δ, then there exists a coloring of the graph by 2Δ colors, where each color class meets each V _i at precisely one vertex. We shall name it the strong 2Δ-colorability conjecture. We prove a fractional version of this conjecture. For this purpose, we prove a weighted generalization of a theorem of Haxell, on independent systems of representatives (ISR’s). En route, we give a survey of some recent developments in the theory of ISR’s. The research of the first author was supported by grant no 780/04 from the Israel Science Foundation, and grants from the M. & M. L. Bank Mathematics Research Fund and the fund for the promotion of research at the Technion. The research of the third author was supported by the Sacta-Rashi Foundation.     

The antioxidant tempamine: in vitro antitumor and neuroprotective effects and optimization of liposomal encapsulation and release

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4(-2)>Cl-1>glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.     

Imaging photoelectron transmission through self-assembled monolayers: the work-function of alkanethiols coated gold

In this paper, we present a new approach for studying the electronic properties of self-assembled monolayers and their interaction with a conductive substrate, the low-energy photoelectron imaging spectroscopy (LEPIS). LEPIS relies on imaging of photoelectrons ejected from a conductive substrate and subsequently transmitted through organic monolayers. Using this method, we measure the relative work-function of alkanethiols of different length on gold substrate, and we are able to follow the changes occurring when the surface coverage is varied. We also computed the work-function of model alkanethiols using a plane-wave density functional theory approach, in order to demonstrate the correlation between changes in the work-function with the monolayer organization and density.     

Why web‐based network monitoring? Leveraging the platform

The increasing use of network monitoring and the growth of the Internet and intranets are converging trends that make IP network infrastructures the logical means of delivering network monitoring, using browser ‐bused clients. Copyright © 1999 John Wiley & Sons, Ltd.