No abstract

Abstract. Platelets in circulation normally do not adhere to resting endothelial cells. However, in response to vascular injury they adhere to stimulated endothelium and thereby play an essential role in hemostasis and thrombosis. Infection with dengue-2 virus can cause illness accompanied by thrombocytopenia and hemorrhage. Increased adherence of platelets to stimulated endothelial cells could contribute to the thrombocytopenia. In this study, adherence of radioisotopically labeled platelets to 1) unstimulated, 2) lipopolysaccharide (LPS)-stimulated, and 3) dengue-2 virusinfected human umbilical vein endothelial cells (HUVEC) was measured in an in vitro assay. Primary HUVEC were cultured in 96-well tissue culture plates in the presence or absence of LPS or dengue-2 virus. These cells were coincubated with 3 H-adenine-labeled fresh platelets for 30 min after which the cells were assayed for adherent platelets. Within 30 min there was maximum adherence of platelets to confluent LPS-stimulated HUVEC (36 ± 4% over controls; P ‫ס‬ 0.005). In comparison, there was a significant increase in adherence to dengue-2 infected HUVEC (78 ± 7%; P Յ 0.001). Additionally, platelet adherence was visualized using fluorescent microscopy. Dengue-2 infection stimulated the HUVEC as monitored by expression of E-selectin. Platelets that adhered to dengue-2 or LPS-stimulated HUVEC were activated as visualized by dual fluorescent probes. These data demonstrate that human platelets adhere to dengue-2 virus-stimulated HUVEC and this interaction could contribute to the thrombocytopenia observed during infection.

Abstract. Mixtures of pre-formed microtubules, polymerized from chicken erythrocyte and brain tubulin, rapidly anneal end-to-end in vitro in standard microtubule assembly buffer. The erythrocyte tubulin segments in annealed heteropolymers can be distinguished by an immunoelectron microscopic assay that uses an antibody specific for chicken erythrocyte betatubulin. An annealing process is consistent with the following observations: (a) Microtubule number decreases while the polymer mass remains constant. (b) As the total number of microtubules declines, the number of heteropolymers, and the number of segments contained in each heteropolymer, increases. (c) The size of the segments determined after annealing and antibody labeling is the same as the original microtubule polymers. (d) Points of discontinuity in the annealing heteropolymers can be observed directly by electron microscopy, and correspond to type-specific polymer domains. The junctions probably represent initial contact points during the annealing process. Microtubule annealing occurs rapidly in vitro and may be significant for determining properties of microtubule dynamics in vivo.I T is generally accepted that the dynamic properties of microtubules, such as the redistribution of microtubule lengths at steady state, can be explained in terms of the association of tubulin dimers with microtubule ends (1-4) rather than by the fragmentation and reannealing of microtubule polymers. Carlier and co-workers (5, 6) proposed a mechanism for length redistribution based on the appreciable differences in the rate constants for the association and dissociation of GTP-tubulin vs. GDP-tubulin with the ends of microtubules. Once the GDP-tubulin cores of small polymers are exposed to solvent, they depolymerize quickly (5, 7), and, due to the low rate of association of GTP-tubulin subunits with microtubules containing GDP-tubulin ends, the conversion back to growing polymers containing GTP-tubulin caps occurs infrequently (8). As the microtubule number decreases, the remaining microtubules with GTP-tubulin caps can lengthen because of an increase in the concentration of the tubulin dimer pool. The result of this activity is fewer but longer polymers. Although several investigators have observed microtubules to undergo changes in length (4, 9), the observations on length redistribution are also consistent with a redistribution mechanism based on microtubule annealing.In studies of the kinetics of actin polymerization, the significance of fragmentation and annealing has long been an area of interest. Experiments by Kawamura and Maruyama (10), Nakaoka and Kasai (11), and Kondo and Ishiwata (12) suggest that small F-actin fragments associate end-to-end to yield longer filaments in a concentration-dependent fashion, and recent models for actin polymerization (13-15) were found to fit the observed assembly kinetics better when terms for fragmentation and annealing of F-actin were included. Nevertheless, annealing of actin filaments or microtubuleshas not yet been directl...

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2-DE and/or MS need to be complemented by different kinds of technologies. The well-known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody-centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label-free detection technologies, human genome-scale numbers of analytes, and increases in analytic and bioinformatic speeds.

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Complete androgen insensitivity syndrome (CAIS), or so-called testicular feminization, results from the lack of androgen action on target organs. Within this syndrome, two major variants have been described. In the first variant, the specific intracellular androgen receptors are undetectable (CAIS, AR-), whereas normal levels of androgen receptors are measured in the second variant (CAIS, AR+). From studies with cultured labial skin fibroblasts of three CAIS, AR+ patients from the same family, we have demonstrated that their androgen receptors present qualitative differences when compared to normal receptors: 1) the apparent affinity constant (Kd) of 5 alpha-dihydrotestosterone for the receptor is higher than normal; 2) the in vitro dissociation rate of the receptor-steroid complex is faster than normal: 3) the cellular androgen receptor is more thermolabile than normal when cells are exposed to superphysiological temperatures; and 4) the relative binding affinity of the androgen receptor for progesterone is greater than normal. These findings suggest the presence of a structural abnormality of the androgen receptor in patients with CAIS, AR+. However, these changes (e.g. slightly decreased affinity of the receptor for 5 alpha-dihydrotestosterone) probably do not explain the total lack of androgen action in these patients. Finally, the present data from this family along with those from the literature suggest the presence of heterogeneity as to the cause of the defect in CAIS, AR+.

This report describes a rapid and efficient method of assessing the survival of human platelets in a rabbit model using flow cytometry. This technique will enable the monitoring in rabbits of human platelets prepared by various preservation protocols.

No abstract