Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.     

Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations

Antibodies are valuable tools in the laboratory and clinic. Antibodies include those secreted by a single clone of B lymphocytes, termed monoclonal antibodies, and those produced by a mixture of various B lymphocyte clones, termed polyclonal antibodies. Both products have become essential instruments in fundamental immunological research, immunohistochemistry, diagnostic testing, and vaccine quality control. Antibody production requires a substantial number of animals, and the animals are subjected to a number of invasive procedures such as antigen injection and blood collection. However, by carefully designing an immunization protocol and by optimizing the immunization response, it is possible to minimize animals' pain and distress while obtaining optimal immune responses. In this article, the critical steps in the production of polyclonal and monoclonal antibodies are described, specifically including selection of the animal species and its age, injection protocol, and ascites tapping. Recommendations are provided for optimizing the immunization response.     

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups

Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and α₂Macroglobulin (α₂M) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and α₂M levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, α₂M plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of α₂M to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.     

Effects of the introduction of in vitro assays on the use of experimental animals in pharmacological research

The introduction of in vitro assays in pharmacological research has led to a reduction in the number of experimental animals used. But what has been the degree of this reduction, and when did it really start? This report describes the events in a medium-sized pharmaceutical company. Analysis of data collected over the last 12 years shows a five-fold reduction in the number of experimental animals used per compound synthesised. Compounds from compound libraries (large collections of randomly-synthesised molecules) that are being assessed for potential bioactivity in 'high-throughput screening' were not included in this analysis. Over the years, the (average) degree of discomfort for the animals in the experiments did not vary much; with variation generally observed from 1.5 to 2.0 (on a scale from 1-6). There was a peak in the discomfort score of experimental mice in 1997, which could be explained by the initiation of arthritis models that were subsequently refined, resulting in a lower degree of suffering. It might be concluded that the introduction of in vitro assays has indeed brought about a significant reduction in the number of experimental animals required to select a good compound (i.e. one that could progress to the preclinical toxicology phase). However, this development appears to have been neutralised by the low survival rate of new chemical entities in clinical studies, leading to a lower number of compounds per annum that actually reach the market place. Put in this 'productivity perspective', the number of experimental animals required to select a marketable drug has not much changed in the last decade.     

Free factor Xa is on the main pathway of thrombin generation in clotting plasma

The effect of a synthetic pentasaccharide that specifically causes the inactivation of factor Xa on the development of prothrombinase activity in human plasma was monitored using four triggers of coagulation: (a) human brain thromboplastin; (b) contact activation; (c) factor X activating enzyme complex; (d) prothrombin activating enzyme complex. Inhibition was similar with the triggers a, b and c. With prothrombinase (d), the inhibition strongly decreased with increasing amounts of factor Va present. This indicates that only free factor Xa is inhibited. Because both the intrinsic pathway (b) and the extrinsic pathway (a) are inhibited by the pentasaccharide, we conclude that free factor Xa plays a rate-limiting role in the pathways, so that there is no reason to postulate the existence of 'supercomplexes' consisting of factors IXa, VIIIa, X(a), Va and prothrombin adsorbed on the same phospholipid particle (intrinsic system) or factor VII(a), X(a), Va and prothrombin adsorbed on tissue thromboplastin (extrinsic system).     

Modulation of autonomic neurotransmission by PGD2: comparison with effects of other prostaglandins in anesthetized cats

Experiments with anesthetized cats were done to study possible roles of different prostaglandins (PGs) in modulating sympathetic neuroeffector transmission. We recorded contractions of the nictitating membrane (n.m.), blood flow in the carotid artery, heart rate and blood pressure, both under control conditions and while stimulating the cut cervical sympathetic nerve. Intra-carotid arterial injection (i.a.) of PGD2 depressed sympathetic transmission to the n.m. without depressing the effects of exogenous norepinephrine (NE). In contrast, PGE2 enhanced the effects of nerve transmission or exogenous NE on the stimulated n.m. PGI2 had similar but shorter effects to PGE2. PGF2 alpha or a stable PGH2 analog, contracted the n.m. smooth muscle with no detected effect on nerve transmission. Carotid blood flow was increased by PGD2, PGE2 and PGI2. PGD2 and PGI2 caused bradycardia that could be blocked by atropine. This ability of PGD2 to modulate autonomic nerve activity is of particular interest because of recent reports that nerve tissue synthesizes PGD2.     

Refinement of polyclonal antibody production by combining oral immunization of chickens with harvest of antibodies from the egg yolk

Polyclonal antibody production in mammals is generally associated with multiple injections of antigens and adjuvants and repeated blood sampling procedures. During the past 20 yr, the use of chickens instead of mammals for this purpose has increased. A major advantage of using birds is that the antibodies can be harvested from the egg yolk instead of serum, thus making blood sampling obsolete. In addition, the antibody productivity of an egg-laying hen is much greater than that of a similar sized mammal. This article focuses on the developments in oral immunization strategies for chickens that combined with the antibodies from the egg yolk, have great potential for active implementation of the three Rs (replacing, reducing, and refining the use of laboratory animals to the extent possible) in polyclonal antibody production schemes.