Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.     

In situ formation of blends by photopolymerization of poly(ethylene glycol) dimethacrylate and polylactide

Blends of cross-linked poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(d,l-lactide) (PLA) were prepared by mixing photoactive PEGDMA (molecular mass: 875 g/mol) and PLA, and subsequently photopolymerizing the mixture with visible light. The effects of PLA molecular mass and mass fraction on the rheological properties of the PEGDMA/PLA mixtures, and on the degree of methacrylate vinyl conversion (DC), as well as blend miscibility, microstructure, mechanical properties, in vitro swelling behavior, and cell responses were studied. PLA-2K (molecular mass: 2096 g/mol) and PLA-63K (molecular mass: 63 000 g/mol) formed miscible and partially miscible blends with cross-linked PEGDMA, respectively. The addition of the PLA-2K did not affect the immediate or post-cure (>24 h) DC of the PEGDMA upon photopolymerization. However, the addition of PLA-63K decreased the immediate DC of the PEGDMA, which can be increased through extending the curing time or post-curing period. Compared to the cross-linked neat PEGDMA and PLA-2K/PEGDMA blends, PLA-63K/PEGDMA blends were significantly stronger, stiffer, and tougher. Both types of blends and the cross-linked PEGDMA swelled when soaked in a phosphate buffered saline (PBS) solution. The attachment and spreading of MCT3-E1 cells increased with increasing PLA-63K content in the blends. The facile and rapid formation of PEGDMA/PLA blends by photopolymerization represents a simple and efficient approach to a class of biomaterials with a broad spectrum of properties.     

Polygonal planforms and phyllotaxis on plants

We demonstrate how phyllotaxis (the arrangement of leaves on plants) and the ribbed, hexagonal, or parallelogram planforms on plants can be understood as the energy-minimizing buckling pattern of a compressed sheet (the plant's tunica) on an elastic foundation. The key idea is that the elastic energy is minimized by configurations consisting of special triads of periodic deformations. We study the conditions that lead to continuous or discontinuous transitions between patterns, state testable predictions, and suggest experiments to test the theory.     

The transforming activity of sonicated Haemophilus influenzae DNA

Summary The inactivation of transforming Haemophilus influenzae DNA by sonication in aqueous solution was investigated. The molecular weight decrease of the molecules is the major factor in DNA inactivation. It impairs strongly the uptake of the DNA by the recipient bacteria, especially when the molecular weight is lower than 3x10⁶ daltons. The uptake of sonicated DNA by the bacterial cells seems not to be further reduced when molecules of about 0.5x10⁶ daltons are submitted to further depolymerisation. However the transforming activity of these molecules is still sensitive to further sonication. The transforming activity of the sonicated DNA is related in the last resort to the intact length of the DNA molecules, at the level of their single-strand structure, available for recombination. Rupture by ultrasound was found to be twice as efficient in reducing transforming activity as a nick induced by pancreatic DNAse.     

Phage display as a powerful tool to engineer protease inhibitors

Since its introduction by Georges Smith some 25 years ago, phage display has proved to be a powerful molecular technique for selecting proteins with desired biological properties from huge libraries. Early on, various protease inhibitor scaffolds were displayed at the surface of filamentous phages to select new inhibitors with shifted specificities and enhanced affinities towards one or more target protease(s). The past two decades have seen a number of natural protease inhibitors subjected to phage display, mostly to shift and increase their inhibitory specificity, but also to explore the molecular mechanisms by which they interact with their cognate enzymes with low or very high selectivity. This review focuses on the major uses of phage display in the field of protein protease inhibitors. The exquisite molecular mechanisms by which natural protease inhibitors prevent unwanted or excessive proteolysis in cells and tissues are also examined along with some of the general principles underlying the way phage display is applied to these molecules.     

A cluster randomised controlled trial of the community effectiveness of two interventions in rural Malawi to improve health care and to reduce maternal, newborn and infant mortality

Background

Paclitaxel-eluting stents decrease angiographic and clinical restenosis following percutaneous coronary intervention compared to bare metal stents. TAXUS Element is a third-generation paclitaxel-eluting stent which incorporates a novel, thinner-strut, platinum-enriched metal alloy platform. The stent is intended to have enhanced radiopacity and improved deliverability compared to other paclitaxel-eluting stents. The safety and efficacy of the TAXUS Element stent are being evaluated in the pivotal PERSEUS clinical trials.

Methods/Design

The PERSEUS trials include two parallel studies of the TAXUS Element stent in single, de novo coronary atherosclerotic lesions. The PERSEUS Workhorse study is a prospective, randomized (3:1), single-blind, non-inferiority trial in subjects with lesion length ≤28 mm and vessel diameter ≥2.75 mm to ≤4.0 mm which compares TAXUS Element to the TAXUS Express² paclitaxel-eluting stent system. The Workhorse study employs a novel Bayesian statistical approach that uses prior information to limit the number of study subjects exposed to the investigational device and thus provide a safer and more efficient analysis of the TAXUS Element stent. PERSEUS Small Vessel is a prospective, single-arm, superiority trial in subjects with lesion length ≤20 mm and vessel diameter ≥2.25 mm to <2.75 mm that compares TAXUS Element with a matched historical bare metal Express stent control.

Discussion

The TAXUS PERSEUS clinical trial program uses a novel statistical approach to evaluate whether design and metal alloy iterations in the TAXUS Element stent platform provide comparable safety and improved procedural performance compared to the previous generation Express stent. PERSEUS trial enrollment is complete and primary endpoint data are expected in 2010. PERSEUS Workhorse and Small Vessel are registered at http://www.clinicaltrials.gov, identification numbers NCT00484315 and NCT00489541.     

Seed coat variation in Glycine Willd. subgenus Glycine (Leguminosae) by SEM

Seed of the genusGlycine Willd. typically exhibits a muriculate appearance resulting from adherence to the true seed coat of the perisperm or inner pod wall layer. Thickened cell walls of the perisperm superimpose a reticulate network on the seed coat, the type of network ranging from alveolate to stellate depending on the shape of the perisperm cells. Tubercles distributed at intervals give the seed its roughened appearance. Seed lacking an attached perisperm appears smooth and shiny. Seed morphology of 62 collections representing the six species of the subgenusGlycine is examined in detail to elucidate inter-and intraspecific variability. Seed perisperm pattern appears to be characteristic for each species, but there are exceptions.Glycine canescens F. J. Herrn. andG. clandestina Willd. seeds possess a reticulate network and tubercles of irregular shape, the perisperm appearing granular inG. clandestina. Seeds ofG. latrobeana (Meissn.) Benth. andG. tabacina (Labill.) Benth. lack a distinct network and have stellate tubercles; the perisperm is granular inG. latrobeana and some plants ofG. tabacina. A few collections ofG. clandestina approachG. tabacina in seed appearance.Glycine tomentella Hayata seeds exhibit a regularly alveolate arrangement, while those ofG. falcata Benth. lack a perisperm layer altogether. Variation in seed coat within a species can usually be linked to differences in chromosome number or some aspect of gross morphology. Diploid collections ofG. tomentella (2n = 40) exhibit recognizable differences in seed morphology compared with tetraploids (2n = 80), coincident with other striking dissimilarities in gross morphology. An incompletely attached perisperm is accompanied by aneuploidy in severalG. tomentella accessions, while other 78 and 38 chromosome aneuploids produce normal seeds.     

Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.     

Use of Multilocus Sequence Typing To Investigate the Association between the Presence of Campylobacter spp. in Broiler Drinking Water and Campylobacter Colonization in Broilers

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.