A novel form of the plasminogen activator inhibitor created by cysteine mutations extends its half-life: relevance to cancer and angiogenesis.

Proteolytic activity driven by urokinase (uPA) is commonly recognized as an important factor in metastasis and angiogenesis. The eradication of unwanted uPA activity expressed by cancer cells results in the inhibition of metastasis and angiogenesis. Development of novel and highly selective uPA inhibitors could, therefore, produce new treatments of cancer. The ultimate goal of this work is the identification of novel and selective inhibitors of uPA suitable for this purpose. We have chosen plasminogen activator inhibitor(s) type 1 (PAI-1), which selectively inhibits the urokinase plasminogen activator (uPA). However, PAI-1 is not a stable molecule and converts itself into the latent form with a half-life in the range of t1/2 = 1-2 h. This conversion is associated with a partial insertion of the reactive loop (P4-P10') into the PAI-1 molecule. In such a conformation, P1-P1' and other sites are not accessible for reaction with uPA. To conquer this hurdle, we have produced several PAI-1 mutants by replacing chosen amino acids with cysteine in the hope of creating disulfide bridges, which could make this insertion more difficult. On the basis of the known structure of active PAI-1, we have identified amino acids that can be substituted with a cysteine residue to produce disulfide bridges linking the top and bottom parts of strands A3 and A5 as well as sites within the helix-D region. We created a total of seven cysteine mutants via point mutation (two to six point mutations), generating possible sites for disulfide bridge formation at the top and bottom parts of A3 and A5, within the helix-D region, or by a combination thereof. Desired mutations were introduced by PCR using appropriate primers. The mutant forms of PAI-1 containing the chitin-binding intein tag were then purified using affinity chromatography wherein the intein tag is cleaved, leaving mutant PAI-1 protein. Cys mutations resulted in proteins with extended half-life of PAI-1 from 2 to >700 h depending on the mutant. Novel PAI-1 were fully functional against uPA and showed activity in the in vitro model of angiogenesis, e.g., in the inhibition of sprout formation. Such prolonged serpin activity, which is therapeutically desired in cancer treatment and Cys-mutated PAI-1, could launch a new class of novel anticancer agents.

Computer simulation of prostate cryoablation--fast and accurate approximation of the exact solution.

OBJECTIVE: Cryosurgery is a minimally invasive cancer treatment that uses liquid nitrogen or supercooled argon to freeze and destroy tumors. To achieve complete ablation of the prostate, we have developed a computer program that can determine treatment effects by calculating iceball formation. This is based on a three-dimensional (3D) model of the patient's prostate constructed from ultrasound images. The program predicts the best set of cryoablation parameters and cryoprobe spatial positions, then displays these parameters in graphical or numerical form. The objective of this work was to improve our prostate cryoablation modeling software by making its partial differential equation (PDE) solver more accurate and faster. MATERIALS AND METHODS: CryoSim, our software package, accepts a set of acquired and processed 3D ultrasound images of the prostate, then models heat diffusion using numerical approximations of the heat equation. RESULTS: We describe the latest version of the CryoSim software, which models cryoablation therapy. Solving the problems of low spatial resolution (now down to a fraction of a millimeter, as compared to 5 mm in the old version) and modeling cryosurgery in a short time (down to few minutes versus hours) provides a platform for proper planning of cryosurgery and a tool for the training of surgeons. CONCLUSION: Changes in the PDE solver algorithm produce more accurate results, leading to an improved visualization of the iceball, with a precision of a few mm and a significant decrease in computation time.

Phase diagrams and solvation forces of a uniaxial ferromagnet in a slit--the double-parabola approach.

We obtain the phase diagrams of a uniaxial ferromagnet enclosed in a slit and analyse the properties of the solvation forces acting between the slit walls. Our analysis is based on the mean-field theory applied to the double-parabola Hamiltonian for a uniaxial ferromagnet in a slit, which allows us to derive several results in analytic form. In particular, the Kelvin law and the law describing the influence of the slit width on the position of the delocalization transition as compared to the wetting transition are derived and the corresponding critical exponent is evaluated. The influence of the thermodynamic state of the system on the properties of the solvation forces, in particular their discontinuous behaviour at coexistence lines, is discussed.

RNA tertiary structure determination: NOE pathways construction by tabu search.

MOTIVATION: Liquid state nuclear magnetic resonance (NMR) spectroscopy has now been well established as a method for RNA tertiary structure determination. Most of the steps involved in the determination of RNA molecules are performed using computer programs. They however, do not apply to resonance assignment being the starting point of the whole procedure. We propose a tabu search algorithm as a tool for automating this step. Nuclear overhause effect (NOE) pathway, which determines the assignment, is constructed during an analysis of possible connections between resonances within aromatic/anomeric region of two-dimensional NOESY spectrum resulting from appropriate NMR experiment. RESULTS: Computational tests demonstrate the superior performance of the tabu search algorithm as compared with the exact enumerative approach and genetic procedure applied to the experimental and simulated spectral data for RNA molecules. AVAILABILITY: The software package can be obtained upon request from Marta Szachniuk.