Protease-containing silicates as active antifouling materials

Biocatalytic silicates, composite materials composed of alpha-chymotrypsin and a silicate prepolymer, were prepared via a two-step polymerization process following solubilization of the enzyme in the polymerization media. This new approach resulted in active and stable composites, and a calculated half-life of over 350 days in aqueous buffer at 30 degrees C. The high stability and activity of this biocatalytic silicate was likely due to the covalent attachment between alpha-chymotrypsin and the silicate matrix. The protease-containing silicate was resistant to fouling by nonselective protein binding, as demonstrated by the dramatically reduced binding of human serum albumin to the silicate material when compared to that of a silicate containing pre-inactivated alpha-chymotrypsin.     

Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.     

Five-color flow cytometric analysis of swine lymphocytes for detection of proliferation,apoptosis, viability,and phenotype

BACKGROUND: The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen-stimulated porcine lymphocytes. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin. RESULTS: Once isolated, PBMCs were stained with the fluorescent membrane intercalating dye, PKH67, and cultured with or without B. hyodysenteriae whole-cell sonicate antigen. Serial samples of nonstimulated and B. hyodysenteriae-stimulated PBMCs were harvested for flow cytometric analysis. Fluorochrome excitation was performed with spatially separated air-cooled argon and red helium neon laser beams. Five-color analysis included signal detection of PKH67 (proliferation), phycoerythrin (cell surface antigen), Texas Red phycoerythrin tandem (cell surface antigen), allophycocyanin (annexin V), and 7-amino-actinomysin D (7AAD; viability). For analysis, gates were set on live (annexin V(-), 7AAD(-)), intact apoptotic (annexin V(+), 7AAD(dim)), and live plus intact apoptotic (annexin V(+/-), 7AAD(dim/-)) cells, and the phenotypes of PBMCs within these populations were determined during the course of the in vitro response. Dead cells (i.e., 7AAD(bright)) were excluded from the analysis. CONCLUSION: Application of this method for the determination of porcine lymphocyte subset proliferation is presented.     

Application of a probabilistic microstructural model to determine reference length and toe-to-linear region transition in fibrous connective tissue

This study shows how a probabilistic microstructural model for fibrous connective tissue behavior can be used to objectively describe soft tissue low-load behavior. More specifically, methods to determine tissue reference length and the transition from the strain-stiffening "toe-region" to the more linear region of the stress-strain curve of fibrous connective tissues are presented. According to a microstructural model for uniaxially loaded collagenous tissues, increasingly more fibers are recruited and bear load with increased tissue elongation. Fiber recruitment is represented statistically according to a Weibull probability density function (PDF). The Weibull PDF location parameter in this formulation corresponds to the stretch at which the first fibers begin to bear load and provides a convenient method of determining reference length. The toe-to-linear region transition is defined by utilizing the Weibull cumulative distribution function (CDF) which relates the fraction of loaded fibers to the tissue elongation. These techniques are illustrated using representative tendon and ligament data from the literature, and are shown to be applicable retrospectively to data from specimens that are not heavily preloaded. The reference length resulting from this technique provides an objective datum from which to calculate stretch, strain, and tangent modulus, while the Weibull CDF provides an objective parameter with which to characterize the limits of low-load behavior.     

Reconstruction of the three-dimensional NMR spectrum of a protein from a set of plane projections

Three-dimensional protein NMR spectra can be obtained significantly faster than by traditional methods by a projection-reconstruction procedure related to X-ray tomography. First, two orthogonal projections are acquired in quick two-dimensional experiments with the evolution parameters t₁ or t₂ set to zero. These projections define a three-dimensional lattice; all cross-peaks must lie on this lattice but not all lattice points are occupied. A third experiment with t₁ and t₂ incremented simultaneously and in a fixed ratio, generates a projection onto a tilted plane and thus establishes the positions of all the cross-peaks unambiguously. This projection-reconstruction technique has been tested on the 500 MHz three-dimensional HNCO spectrum of ubiquitin.