The adsorption kinetics of purified fibrinogen to unmodified and aminopropylsilane-modified quartz glass surfaces were studied under pseudo-first order (binding-unit excess) conditions by the total internal reflection fluorescence (TIRF) method. Fluorescence in the adsorbed protein layer (350 nm) was excited by the evanescent wave at 285-290 nm. In order to reduce and possibly eliminate the influence of mass transfer on the kinetics of fibrinogen adsorption, a novel protein adsorption chamber containing a cone-and-plate rheometer with total internal reflection technology was employed. The aim of the study was to obtain critical shear rates, at which the adsorption rate of fibrinogen became independent of diffusion. Therefore, shear rates were varied between 0 and 7200 s-1 at initial fibrinogen concentrations of c9 = 4.7 and 17.7 micrograms mL-1. The adsorption rate of fibrinogen increased 5-17-fold, depending on the surface, as the critical shear rate was approached. Above the critical shear rates the kinetic data of fibrinogen adsorption could be fitted at c9 = 4.7 micrograms mL-1 to a single exponential function, indicating the predominance of a single binding step with a half-life of ca 20 s. At the higher initial concentration of c9 = 17.7 micrograms/mL-, however, a significant deviation from the single exponential behavior was observed in the first 10 s of the adsorption reaction, indicating a very fast initial event with a half-life of ca 5 s in addition to a slower binding reaction with a half-life of ca 35 s. Thus the novel TIRF rheometer can resolve kinetics down to half-lives of 5 s and possibly even lower.

Adsorption↗

PubMedDate not supplied

Conjugation of unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, to monoclonal antibody CC49 by an improved active ester protocol.

For the conjugation of the trihydroxamate bifunctional chelating agent N-[tris[2-[[N-(benzyloxy)amino]-carbonyl]ethyl]methyl]succinamic acid (trisuccin, 1) to antibodies, we originally used the corresponding 2,3,5,6-tetrafluorophenyl active ester followed by the postconjugation removal of the benzyl protecting groups by catalytic hydrogenation. It was of interest to us to design a conjugation protocol capable of incorporating deblocked hydroxamates into peptides and proteins. Reported procedures that were expected to be compatible with the functionalities present in trisuccin were used with no success, as judged by the lack of ability of the products to radiolabel with 188Re. A simple conjugation method was then developed utilizing the o-nitrophenol (ONP) activated ester of the unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, 3, which eliminates the need for the postconjugation deblocking. An assay for indirect estimation of the active ester content, based on the concentration of its decomposition byproduct, ONP-OH, was developed. Comparison of the indirectly estimated concentrations with those obtained directly from purified products showed > 90% accuracy for this assay. This procedure has the advantage of rapidly using the unpurified active ester, eliminating the possibilities of its decomposition through solvolysis or self-condensation by the unprotected hydroxamate functions. A colorimetric assay was developed for estimation of the number of ligands per molecule of protein. This assay and the fact that all conjugates consistently radiolabeled with 188Re show that this procedure conjugated the unprotected hydroxamate ligands to the CC49 monoclonal antibody. These results indicate the potential applicability of this technique to conjugation of unprotected hydroxamate derivatives with other proteins and peptides.

Antibodies, Monoclonal↗

PubMedDate not supplied

Rural ambulance service in West Virginia.

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Ambulances↗

PubMedDate not supplied

Is Hughes-Stovin syndrome Behçet's disease?

Hughes-Stovin syndrome (HSS) is a rare clinical disorder, which has been described as the presence of pulmonary artery aneurysm in the setting of systemic thrombosis. The term "Incomplete Behçet's Disease" has also been used to describe this syndrome due to the clinical and histopathological similarities between Behçet's disease and HSS. Indeed, pulmonary involvement can be indistinguishable between these two conditions of unknown pathophysiology. We describe an HSS patient who presented with a recurrent pulmonary artery aneurysm, review the clinical and pathological manifestations of HSS, discuss its similarities to Behçet's disease, and finally make the argument that HSS is in fact Behçet's disease.

Adult↗